JP2001526914A - Human / regulatory protein - Google Patents

Abstract

  (57) [Summary] The present invention provides human regulatory proteins (HRGPs) and polynucleotides that identify and encode HRGPs. The present invention also provides expression vectors, host cells, agonists, antibodies, and antagonists. The present invention also provides methods for diagnosing, treating, and preventing a disease associated with HRGP expression.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention relates to nucleic acid and amino acid sequences of important human regulatory proteins in disease, and diagnosis of diseases associated with cell proliferation, prevention, it relates to use of these sequences in the treatment.

[0002] Growth and differentiation BACKGROUND OF THE INVENTION cells play a role in structural role or cellular metabolism, response to their environment by changing the involvement in the development of the organism, and cellular gene expression I do. Cell function is regulated by the timing and amount of expression resulting from thousands of genes. Regulation of expression is important to conserve energy and prevent the synthesis and accumulation of intermediates such as untranslated RNA and incomplete or inactive proteins.

[0003] Regulatory protein molecules are essential for regulating gene expression. These molecules regulate the activity of each gene or group of genes in response to various induction mechanisms of the cell or organism, and whether transcription is initiated, enhanced, or suppressed. By acting as a transcriptional regulator, splicing of the transcript occurs when directed in a particular cell or tissue. Although regulatory molecules interact with short stretches of DNA distributed throughout the genome, most gene expression is regulated within the open reading frame of the expressed gene or near the transcription start site. Stretches of DNA that can be regulated may be single and interact with only one protein, or may require several proteins to function as part of a complex to regulate gene expression.

[0004] The double helical structure and repeats of DNA form external features that can be recognized by regulatory molecules. These external features include hydrogen bonding donor and acceptor groups, hydrophobic patches, major and minor grooves, and regulatory repeat stretches that cause individual bends in the helix. Such a characteristic portion serves as a recognition site for the binding of a regulatory protein. Generally, these recognition sites are 2
Although less than 0 nucleotides in length, multiple sites may be adjacent to each other, each of which may confer regulation on a single gene. Hundreds of these recognition sites have been identified, each of which is recognized by a different protein or protein complex that performs gene regulation.

[0005] A regulatory protein molecule or complex is an open reading frame (ORF)
A specific nucleotide sequence of the upstream (5 'end) untranslated region preceding the first translated exon of the ORF; a specific nucleotide sequence of an intron junction that occurs between many exons of the ORF; Recognizes and binds to a particular nucleotide sequence in the downstream (3 'end) untranslated region. The surface features of the regulatory molecule are broadly complementary to the surface features of the double helix. The individual bonds between the protein (s) and the helix can be relatively weak bonds (hydrogen bonds, ionic bonds, and / or hydrophobic interactions), but due to the multiple junctions between the protein and DNA, A very strong interaction occurs.

Family of Regulatory Molecules Many regulatory molecules incorporate DNA binding structural motifs containing either an α-helix or a β-sheet and bind to the major groove of DNA. The seven structural motifs common to regulatory molecules are the helix-turn-helix structure, homeodomain, zinc finger, steroid receptor, β-sheet, leucine zipper, and helix-loop-helix (HLH) structure.

The helix-turn-helix motif is constructed from two α-helices connected by a short chain of amino acids at a certain angle. The more abundant carboxy-terminal helix is the recognition helix. This is because it fits into the main groove of DNA. The amino acid side chains of this helix recognize the specific DNA sequence to which the protein binds. The structure of the other parts varies widely between regulatory proteins with this motif. The helix-turn-helix structure is not stable without other parts of the protein and does not bind to DNA without other peptide regions that confer stability. Other peptide regions also interact with the DNA, thereby increasing the number of unique sequences that the helix-turn-helix structure can recognize.

[0008] Many sequence-specific DNA binding proteins are, in fact, symmetrical dimers, consisting of two very similar halves of a DN that are also symmetrically organized.
Binds to the A sequence. This structure allows the monomers of each protein to be similarly D
It becomes possible to interact with the NA recognition site, and the number of binding portions with DNA is doubled. By doubling the number of binding parts in this way, the binding affinity is greatly enhanced only by doubling the free energy of interaction. The DNA to which the helix-turn-helix motif always binds is in the form of B-DNA.

[0009] Homeodomain motifs are found on a special group of helix-turn-helix structures encoded by homeotic (homologous heterogenous) selector genes. It is so named because the proteins encoded by these genes regulate developmental switches. For example, mutations in these genes convert one part of the body to another in Drosophila. These genes were found in all eukaryotes examined. The helix-turn-helix regions of different homeodomains are not always identical in sequence, but are always surrounded by the same structure, and the motif is always present in the same form in DNA. This helix-turn-helix structure is stable by itself and can bind to DNA when isolated as well. The helix in the homeodomain is usually longer than the helix in most HLH regulatory proteins. The portion of the motif that interacts almost directly with DNA differs between these two families (eg, Pabo, CO and RT Sauer (
1992) Ann. Rev. Biochem. 61: 1053-1095).

A third motif, called the zinc finger motif, incorporates a zinc molecule into an important part of the protein. Proteins belonging to this family often have a tandem repeat of a 30 residue zinc finger motif, including the sequence pattern Cys-X2 or 4-Cys-X12-His-X3-5-His. Each of these regulatory proteins has an alpha helix and an antiparallel beta sheet. Two histidines in the α-helix and two cysteines near the turn in the β-sheet interact with zinc ions. This zinc ion keeps the α helix and the β sheet close to each other. The bond to DNA is formed by arginine preceding the α-helix and the second, third and sixth residues in the α-helix.
By changing the number of zinc fingers, the specificity and strength of the binding interaction can be changed.

[0011] Steroid receptors are a family of regulatory proteins that include receptors for steroids, retinoids, vitamin D, thyroid hormone, and other important compounds. The DNA binding domain of these proteins contains about 70 residues, eight of which are conserved cysteines. The steroid receptor motif consists of two spherical α-helices which are orthogonal to each other and have a spherical shape. Each helix has a zinc ion that holds a peptide loop to the N-terminus of the helix. The first helix fits into the major groove of the DNA, with the side chain contacting the base edges of the DNA. Steroid receptor proteins, like helix-turn-helix proteins, form dimers that bind to DNA. The second helix of each monomer is
It binds to the phosphate group of the DNA backbone, which also serves as a boundary for dimerization.
There may be multiple options for heterodimerization, creating other mechanisms for the regulation of multiple genes.

Another family of regulatory proteins includes a motif consisting of a double-stranded antiparallel β-sheet that functions in recognition in the major groove of DNA. The exact DNA sequence recognized by this motif is determined by the amino acid sequence of the β-sheet from which the side chains extend and join the DNA. In the example of a β-sheet in two eukaryotes, the regulatory protein forms a tetramer when bound to DNA.

[0013] The leucine zipper motif generally forms a dimer, with 30 to 40 residues each forming a short coiled-coil structure, in which two α-helices, each derived from one of the monomers, combine. Having. Interactions between the side chains of hydrophobic amino acids, often on the 7-fold leucine, extending from one side chain of each helix,
The helix is held together. In the later part of the structure, the helix separates and each basic region joins the major groove of the DNA. Proteins with this motif can form either homodimers or heterodimers, extending the specific combinations available for regulation of expression.

Another important motif is the short α linked by a loop to a long α helix
It is a helix-loop-helix (HLH) structure consisting of a helix.
The loop is flexible and allows the two helices to fold back on each other. The α-helix can bind to DNA as well as to the HLH structure of another protein. The second protein may be the same as the first protein, ie, the protein that produces a homodimer, or a different protein, ie, a protein that produces a heterodimer. Some HL
Although H monomers do not have enough α-helices to bind to DNA, these monomers can form heterodimers that can affect certain regulatory proteins.

To date, hundreds of regulatory proteins have been identified and more are being characterized in various organisms. Most regulatory proteins have at least one of the aforementioned common structural motifs that mediate conjugation with DNA.
Having one. However, some important regulatory proteins, such as the p53 tumor suppressor gene, do not share their structure with other known regulatory proteins. Changes in known motifs and new motifs have been characterized and are still being characterized (see, for example, Faisst. S. and S. Meyer (1992) Nucl. Acids Res. 20: 3-26). .

[0016] Although the binding of DNA to regulatory proteins is very specific, the exact DNA sequence that a particular regulatory protein favors, or the primary structure of a regulatory protein for a particular DNA sequence, is unpredictable. Thus, the interaction between DNA and regulatory proteins is not limited to the motifs described above. Other domains of the protein often form important junctions with DNA, and accessory proteins can convert specific protein complexes into activators or repressors or prevent binding (See, for example, Alberts, B. et al. (1994
) Molecular Biology of the Cell , Garland Publishing Co, New York, NY pp.
401-474).

Diseases and Diseases Associated with Gene Regulation The causes of neoplastic growth in humans are often due to problems in gene regulation. The growth of malignant cells may be due to an excess of transcriptional activators or a deficiency of inhibitors or suppressors (eg, Cleary, ML (1992) Cancer Surv. 15: 89-1.
04). The gene fusion creates a chimeric locus with a switch domain, which can interrupt proper activation of the target gene by the chimera.

The response of a cell to an infection or trauma is beneficial when gene expression is appropriate. However, inadequate or inadequate regulation of gene expression can lead to hyperregulatory response or other imbalances, which can lead to significant tissue and organ damage. This injury has been well documented in immunological responses to allergens, heart attacks, strokes, and infections (eg, Harrison's Principles of Internal Medicine , 13th ed., (1994) McGraw Hill, Inc. and Tetron). Data Sy
stems Software). In addition, the accumulation of somatic mutations and increased inability to modulate cellular responses has been associated with the prevalence of osteoarthritis and the development of other diseases associated with aging.

The discovery of novel human regulatory protein molecules, and polynucleotides encoding those molecules, which are important in disease development, has led to the diagnosis, treatment, and prevention of diseases associated with cell proliferation, particularly the immune response and cancer. The need in the art is met by providing useful new compositions.

( Summary of the Invention ) The present invention relates to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, providing a substantially purified human regulatory protein (HRGP) having an amino acid sequence selected from the group consisting of: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. .

[0021] The present invention further provides an isolated and substantially purified polynucleotide encoding HRGP. In certain embodiments, the polynucleotide comprises SEQ ID NO:
13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:
22, having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 23 and SEQ ID NO: 24.

[0022] In addition, the present invention relates to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9
A polynucleotide or a fragment thereof that hybridizes with any of the polynucleotides encoding HRGP selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

The present invention further provides a polynucleotide comprising a molecule complementary to any one of the polynucleotides encoding HRGP, or a fragment thereof. In another embodiment,
The present invention provides SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16
SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, or fragments thereof. A composition comprising an isolated and purified polynucleotide comprising a sequence is provided.

Further, the present invention relates to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,
Provided is an expression vector comprising at least a fragment of any one of the polynucleotides selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24. In another embodiment, the expression vector comprising the polynucleotide is included in a host cell.

The present invention also relates to a method for producing a polypeptide or a fragment thereof, comprising: (a) an expression vector containing at least a fragment of a polynucleotide sequence encoding HRGP under conditions suitable for expression of the polypeptide; Culturing the host cell containing (b)
And b) recovering the polypeptide from the culture medium of the host cell.

The present invention also provides a pharmaceutical composition comprising substantially purified HRGP together with a suitable pharmaceutical carrier.

The present invention also provides a purified HRGP antagonist. In one embodiment, the invention provides a purified antibody that binds to HRGP.

[0028] The present invention further provides a purified agonist of HRGP.

The present invention also relates to a method for treating or preventing cancer associated with a decrease in the expression or activity of HRGP, which comprises administering to a patient in need of such treatment an effective amount of a pharmaceutical composition containing HRGP. A method for treating or preventing a cancer associated with decreased expression or activity of HRGP, comprising the steps of:

The present invention is also a method for treating or preventing a cancer associated with an increase in the expression or activity of HRGP, which comprises administering to a patient in need of such treatment an effective amount of a pharmaceutical composition comprising HRGP. A method for treating or preventing a cancer associated with increased expression or activity of HRGP, comprising the steps of:

The present invention is also a method of treating or preventing an immune response associated with increased HRGP expression or activity, comprising administering to a patient in need of such treatment an effective amount of a pharmaceutical composition comprising HRGP. A method for treating or preventing an immune response associated with increased HRGP expression or activity, comprising the steps of:

The present invention also relates to a method for stimulating cell proliferation, comprising the steps of:
A method of stimulating cell proliferation, comprising the step of administering an effective amount of

The present invention also provides a method for detecting a nucleic acid sequence encoding a human / regulatory protein in a biological sample, the method comprising: (a) detecting a nucleic acid sequence in the biological sample;
A step of hybridizing a polynucleotide sequence complementary to a polynucleotide encoding RGP to detect a hybridization complex, and (b) a step of detecting the hybridization complex, wherein the hybridization complex is detected. The presence of the nucleic acid sequence in the biological sample correlates with the presence of the nucleic acid sequence encoding the human regulatory protein in the biological sample. To provide a method for detecting

In addition, the present invention relates to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.
, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9,
Provided is a microarray comprising at least one fragment of a polynucleotide encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

The present invention also provides a method for detecting the expression level of a nucleic acid encoding a human / regulatory protein in a biological sample, wherein the nucleic acid sequence of the biological sample comprises:
Hybridizing a complementary polynucleotide to form a hybridization complex, and detecting the hybridization complex to express a nucleic acid sequence encoding a human / regulatory protein in the biological sample. Determining the expression level of a nucleic acid encoding a human / regulatory protein in a biological sample. In a preferred embodiment, the nucleic acid sequence in the biological sample is amplified and labeled by polymerase chain reaction before the step of performing hybridization.

[0036] (Embodiment of the invention) protein of the present invention, nucleotide sequences, and before describing how this invention is not limited to the particular methodology disclosed herein, protocols, cell lines, limited vectors, and reagents It should be understood that they can be varied in many ways. In addition, the terminology used in this specification is used for the purpose of describing specific examples only, and is intended to limit the scope of the present invention which is limited only by the description of the claims. It should also be understood that it was not done.

In this specification and the claims, “an” and “the” (a) denote a singular.
It should be noted that what is described as has a plural meaning unless the context clearly indicates otherwise. Thus, for example, reference to "a certain host cell" includes a plurality of such host cells, and "a certain antibody" includes one or more types of antibodies and well-known to those skilled in the art. And its equivalents.

[0038] All technical and scientific terms used herein are, unless otherwise defined,
It has the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs. Although any methods or materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, vectors, arrays and methodology reported in the literature that may be used in the context of the present invention. Part of the specification. Also, no disclosure of this specification should be construed as an admission that such disclosure in the present invention does not precede the prior art.

( Definition ) As used herein, HRGP refers to any species, specifically bovine, ovine, porcine,
Substantially purified HRGP derived from any of natural, synthetic, semi-synthetic, or recombinant, derived from mammals such as mice, horses, and preferably humans, etc.
Is the amino acid sequence of

As used herein, the term “agonist” refers to HRGP when bound to HRGP
It is a molecule that enhances the effect of or increases the duration of the effect. Agonists may include proteins, nucleic acids, carbohydrates, and any other molecule that binds to HRGP and modulates its effects.

As used herein, “allele” or “allele sequence” is an allelic form of a gene encoding HRGP. An allele results from mutation of at least one position in a nucleic acid sequence, resulting in a mutated mRNA or polypeptide.
The structure or function of A or the polypeptide may or may not change. A given intact gene or recombinant gene may have no, one, or multiple allelic forms. Mutations that result in common alleles are due to natural deletions, additions and substitutions of nucleotides. Each of these types of mutations can occur alone or simultaneously with other mutations, once or more than once in a given sequence.

As used herein, a “mutated” nucleic acid sequence that encodes HRGP, including substitutions, insertions and deletions of different nucleotide residues, and consequently encodes the same or a functionally equivalent HRGP. It will be a polynucleotide. This definition includes H
Polymorphisms having loci other than the normal chromosomal locus of the polynucleotide sequence encoding RGP, polymorphisms resulting from inappropriate or unexpected hybridization with alleles, and specific oligos of the polynucleotide encoding HRGP It contains polymorphisms that are easily detectable or difficult to detect using nucleotide probes. The encoded protein can also be "mutated", including deletions, insertions and substitutions of amino acid residues which produce a silent mutation and result in a functionally equivalent HRGP. Intentional amino acid substitutions can be made by HRG
As long as the biological activity of P is retained, it can be generated based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and also the similarity with respect to amphipathicity of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids having an uncharged polar head group with near hydrophilicity include leucine. Glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.

As used herein, “amino acid” or “amino acid sequence” is an oligopeptide, peptide, polypeptide, or protein sequence, or any fragment thereof, and is a naturally occurring molecule or a synthesized molecule. . The HRGP fragment preferably has a length of about 5 to about 15 amino acids and retains the biological or immunological activity of HRGP. Here, when "amino acid sequence" refers to the amino acid sequence of a naturally-occurring protein molecule, "amino acid sequence" or similar terms refer to the complete, original form of the amino acid sequence associated with the protein molecule described herein. It is not used to limit the amino acid sequence as it is.

As used herein, “amplification” refers to the generation of additional copies of a nucleic acid sequence, usually performed using the polymerase chain reaction (PCR) method well known to those skilled in the art (Dieffenbach, CW and GS Dveksler (1995) PCR Primer. A Laboratory Manual , Cold Spring Harbor Press, Plainview, NY).

As used herein, the term “antagonist” refers to a molecule that, when bound to HRGP, reduces the magnitude of the biological or immunological effect of HRGP or shortens the duration of the effect. It is. Antagonists include proteins, nucleic acids, carbohydrates, antibodies, or other molecules that reduce the effects of HRGP.

As used herein, the term “antibody” refers to an intact antibody molecule, as well as fragments thereof that can bind antigenic determinants, such as, for example, Fa, F (ab ′) ₂ , and Fv fragments. I do. Antibodies that bind to HRGP polypeptides can be produced by using the entire polypeptide or a fragment thereof containing the small peptide of interest as the antigen for immunization. The polypeptide or peptide used to immunize the animal may be derived from translation of RNA or may be chemically synthesized,
If necessary, it can be linked to a carrier protein. Commonly used carriers that chemically bind to the peptide include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). An animal (eg, mouse, rat, or rabbit) is immunized with the conjugated peptide.

As used herein, the term “antigenic determinant” is a fragment of a molecule (ie, an epitope) that associates with a particular antibody. When a host animal is immunized with a portion, or fragment, of a protein, various regions of the protein can trigger the production of antibodies that specifically bind to a given region or three-dimensional structure on the protein. Such a region or structure is called an antigenic determinant. Antigenic determinants can compete with the original antigen (ie, the immunogen used to elicit the immune response) for binding to the antibody.

As used herein, the term “antisense” is a composition that includes a nucleotide sequence that is complementary to a specific DNA or RNA sequence. The term "antisense strand"
Used in the sense of a nucleic acid strand complementary to the "sense" strand. Antisense molecules include peptide nucleic acids, and antisense molecules can be created by any method, including synthesis and transcription. Once introduced into the cell, the complementary nucleotide combines with the natural sequence created by the cell to form a duplex, which inhibits further transcription and translation. The expression "minus (-)" is used in the sense of the antisense strand,
“Plus (+)” is sometimes used to mean the sense strand.

As used herein, the term “biologically active” is a protein that has a structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, "immunologically active" refers to a natural, recombinant, or synthetic HRGP, or oligopeptide thereof, that elicits a specific immune response in suitable animals or cells and binds to a specific antibody. Ability.

As used herein, the term “complementary” or “complementarity” refers to the spontaneous binding of polynucleotides by forming base pairs under acceptable salt concentrations and temperature conditions. For example, the sequence "AGT" binds to the complementary sequence "TCA". The complementarity between two single-stranded molecules is "partial", in which only some nucleic acids bind, or completely if there is complete complementarity between the single-stranded molecules. It can be complementary. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that depend on the binding of nucleic acid strands and in the design and use of peptide nucleic acid (PNA) molecules.

As used herein, the term “composition containing a predetermined polynucleotide sequence” refers to any substance containing a predetermined polynucleotide sequence. The composition may be in the form of a powder formulation or an aqueous solution. A polynucleotide encoding HRGP, for example, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:
A composition comprising 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, or a fragment thereof, is a hybridization probe Can be used as The probe can be stored in a lyophilized state and can be conjugated to a stabilizing agent such as a carbohydrate. In hybridization, the probe can be dispersed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, SDS) and other substances (eg, Denhardt's solution, milk powder, salmon sperm DNA, etc.).

As used herein, “consensus” refers to resequencing to separate unwanted bases and extending them in the 5 ′ and / or 3 ′ direction using XL-PCR ^™ (Perkin Elmer, Norwalk, Conn.). Or a computer program for combining fragments (eg, GELVIEW ^™ Fragment Assembly System)
, GCG, Madison Wis.), And is a nucleic acid sequence derived by combining overlapping sequences of two or more Incyte clones. Consensus sequences may be created by both extension and combination.

As used herein, the expression “correlated with the expression of a polynucleotide” means that the presence of ribonucleic acid is detected by analysis by the Northern method, and the presence of mRNA encoding HRGP in the sample. And thus correlate with the expression of transcripts from the gene encoding the protein.

The term “HRGP” refers to a human polypeptide, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: : 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12
Refers to any or all of

As used herein, a “deletion” is a change in the nucleotide or amino acid sequence such that one or more nucleotides or amino acid residues are missing.

As used herein, the term “derivative” means a nucleic acid encoding HRGP or a nucleic acid complementary thereto or a chemically modified HRGP encoded.
Examples of such modifications include hydrogen to alkyl, acyl, or amino groups. Nucleic acid derivatives encode polypeptides that retain the biological or immunological function of the intact molecule. A derivative polypeptide retains at least one of the biological or immunological functions of the original polypeptide, and is glycosylated,
Modified by PEGylation or some other process.

As used herein, the term “homology” refers to a degree of complementarity. the term"
“(Sequence) identity” can be replaced by the term “homology”. Partially complementary sequences that at least partially inhibit the same sequence from hybridizing to the target nucleic acid are referred to as "substantially homologous." Inhibition of the hybridization between the completely complementary sequence and the target sequence is determined by using a hybridization assay (Southern or Northern blot, solution hybridization, etc.) under conditions of reduced stringency. You can find out. A substantially homologous sequence or probe will compete for, and inhibit, binding (ie, hybridization) with a sequence or probe that is completely homologous to the target sequence under conditions of low stringency. This does not mean that low stringency conditions allow for non-specific binding. This is because low stringency conditions require that the binding of the two sequences to each other be a specific (ie, selective) interaction. The absence of non-specific binding can be determined by using a second target sequence that has no partial degree of complementarity (ie, less than about 30% homology or identity). In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.

The phrase “percent identity” or “% identity” is the percentage of sequence similarity when comparing two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, for example, by using the MegAlign program (DNASTAR, Inc., Madison WI). This MegAlign ^™ program
Different methods, such as the cluster method (eg Higgins, DG and P
An alignment of two or more sequences can be made according to .M. Sharp (1988) Gene 73: 237-244). In the algorithm of the cluster method, a sequence group is divided into clusters (collections) by checking the distance between both sequences for all pairs of sequences. This cluster group is aligned for each pair, and then the group is aligned. The percent similarity between two amino acid sequences, for example, sequence A and sequence B, is (length of sequence A-number of gap residues in sequence A-number of gap residues in sequence B) / (sequence A and sequence B Total number of residue matches between B) × 100. Cases where the similarity difference between the two amino acid sequences is low or not are not included in the percent similarity calculation. The percent identity between nucleic acid sequences can also be determined by other well-known methods, such as the Jotun Hein method (eg, Hein J. (1990) Met
hods Enzymol. 183: 626-645). Identity between sequences can also be determined by other well-known methods, for example, by changing hybridization conditions.

“Human artificial chromosomes” (HACs) may comprise DNA sequences of about 10 kb to 10 mb in size, and are linear miniatures containing all the elements necessary for stable division and maintenance of split chromosomes Chromosome (Harrington, JJ et al. (1997) Nat Genet.
15: 345-355).

As used herein, the term “humanized antibody” is an antibody molecule in which the amino acid sequence of the non-antigen binding region has been modified so as to be closer to a human antibody while retaining its original binding ability.

As used herein, the term “hybridization (hybridization)” is the process by which a nucleic acid strand binds to a complementary strand through base pairing.

As used herein, the term “hybridization complex” is defined as the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases.
A complex formed by two nucleic acid sequences. The hybridization complex is
Either it is formed in solution (for example, in the case of C ₀ t or R ₀ t analysis), or the nucleic acid is comprised of one nucleic acid present in solution and one solid support (eg, a cell, paper on which the cells or the nucleic acid are immobilized, a membrane, etc.). , A filter, a pin, or another nucleic acid immobilized on a glass slide or other suitable substrate).

“Immune response” can refer to a condition associated with inflammation, trauma, an immune disease, or an infectious or genetic disease. These conditions can be characterized by the production of various factors that activate cells and the whole body's defense system, such as cytokines, chemokines, and other signaling molecules.

As used herein, the term “insertion” or “addition” refers to a nucleotide sequence or amino acid sequence that adds one or more nucleotides or amino acid residues, respectively, as compared to a naturally occurring molecule. Refers to the change.

As used herein, the term “microarray” is an array of individual polynucleotides or oligonucleotides on a substrate at high density. Substrates include, for example, paper, nylon or other types of membranes, filter paper, chips, glass slides,
Or any other suitable solid support.

As used herein, the term “modulation” is a change in the activity of HRGP. For example,
Modulation may increase or decrease the activity of the protein, alter the binding properties, or alter other HR
Changes in the biological, functional, and immunological properties of GP result.

As used herein, “nucleic acid sequence” refers to oligonucleotides, nucleotides, or polynucleotides, and fragments thereof, single-stranded or double-stranded, and is of sense or antisense strand, of genomic origin or Synthetic DNA or RNA, peptide nucleic acid (PNA), or DNA-like or RNA-like substances. In this context, a “fragment” is a nucleic acid sequence that is 60 nucleotides or more in length, most preferably a fragment that is 100 nucleotides or more or 1000 or more nucleotides, and 10,000 nucleotides or more.

As used herein, the term “oligonucleotide” is a nucleic acid sequence that can be used in a PCR amplification or hybridization assay, or microarray, and is about 6 or more nucleotides in length, up to about 60 nucleotides, preferably Refers to those that are 15 to 30 nucleotides, more preferably 20 to 25 nucleotides. As used herein, the term "oligonucleotide" is substantially synonymous with the terms "ambrimer", "primer", "oligomer", and "probe" as generally defined in the art.

As used herein, “peptide nucleic acid” (PNA) refers to an antisense molecule, ie, an antigenic agent, comprising an oligonucleotide of at least 5 nucleotides in length attached to the peptide backbone of amino acid residues terminating in lysine. I do. Terminal lysine confers solubility to this material. PNAs can preferentially bind to complementary single-stranded DNA or RNA to stop transcript elongation, and extend their life in cells by polyethylene glycolating PNA (eg, Nielsen, PE et al. (1993) Anticancer Drug Des. 8: 53-63).

As used herein, in the context of a protein, for example, “a portion of a given protein”
The term "part (or part)" is a fragment of the protein. The size of this fragment is from the size of five amino acid residues to the total number of amino acids minus one.
It can range up to a size consisting of a number of amino acid residues. Therefore, "HRG
A protein comprising at least a portion of the amino acid sequence of P "includes full-length HRGP and fragments thereof.

As used herein, the term “sample” is used in its broadest sense. Biological samples suspected of containing the HRGP-encoding nucleic acid or fragment thereof or HRGP itself include body fluids, extracts from chromosomes, organelles or cell membranes isolated from cells, cells, ( In solution or attached to a solid support)
Genomic DNA, RNA, or cDNA, tissue, tissue prints (tissue p
rint) Others are included.

As used herein, the term “specific binding” or “specifically binds” is the interaction of a protein or peptide with agonists, antibodies, and antagonists. This interaction depends on the presence of specific structures (ie, antigenic determinants, or epitopes) on the protein that are recognized by the binding molecule. For example, if the antibody is specific for epitope "A", then in a reaction involving labeled "A" and the antibody, the presence of a protein containing epitope A (i.e., unbound, unlabeled A) is present. , The amount of labeled A bound to the antibody is reduced.

As used herein, the term “stringent (stringent) conditions” refers to conditions that permit hybridization between a polynucleotide sequence and a claimed polynucleotide sequence. Appropriate levels of stringent conditions can be determined, for example, by the concentration of salt or formamide in the prehybridization and hybridization regions, or the hybridization temperature, and are well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or increasing the hybridization temperature.

For example, hybridization under high levels of stringent conditions can occur at about 50% formamide concentration at about 37 ° C. to 42 ° C. Hybridization under low levels of stringent conditions is about 30 ° C.
It can occur at a formamide concentration of about 35% to 25% at a temperature of ３５35 ° C. Specifically, hybridization under high stringency conditions is 50%
Formamide concentration of 5X SSPE, 0.3% SDS, and 200 μg / ml
At 42 ° C. using denatured and denatured salmon sperm DNA. Hybridization under conditions of low stringency can occur when the temperature is reduced to 35 ° C. and the formamide concentration is 35% under the conditions described above. The temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Changes in the above temperature ranges and conditions are well known in the art.

As used herein, the term “substantially purified” refers to a substance that has been removed from its natural environment and isolated or separated so that it has more than 60% of its other constituents naturally associated with it. Preferably, it is a nucleic acid or amino acid sequence from which 75% or more, most preferably 90% or more, has been removed.

As used herein, “substitution” refers to replacing one or more nucleotides or amino acids with different nucleotides or amino acids, respectively.

As used herein, “transformation” refers to the process by which foreign DNA enters and changes a recipient cell. This process can occur under natural or artificial conditions using a variety of well-known methods. Transformation can be performed by known methods for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. This method is selected based on the type of host cell to be transformed and includes, but is not limited to, a method of infecting a virus, a method using electroporation, lipofection, and a method using a particle gun. There can be.
Such "transformed" cells include plasmids in which the inserted DNA is either autonomously replicating or stably transformed cells capable of replicating as part of the host chromosome. is there. Also, some of these cells undergo transient expression of the inserted DNA or RNA for a limited time.

As used herein, a “variant” of HRGP is an amino acid sequence in which one or more amino acids have been changed. This variant may contain "conservative" changes,
In the case of this conservative change, the substituted amino acid will have similar structural and chemical properties, such as when leucine is replaced by isoleucine. In rare cases, a variant may change "non-conservatively," for example, replacing glycine with tryptophan. Similar minor changes include amino acid deletions or insertions, or both. Any amino acids that can be substituted, inserted, or deleted without impairing biological or immunological activity,
For example, it can be determined using a well-known computer program such as DNASTAR software.

( Invention ) The present invention collectively refers to HRGP, and individually refers to HRGP-1, HRGP-2,
HRGP-3, HRGP-4, HRGP-5, HRGP-6, HRGP-7, H
RGP-8, HRGP-9, HRGP-10, HRGP-11, and HRGP-
A polynucleotide encoding HRGP, a human regulatory protein designated as
SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24), and the discovery of the use of these compositions for the diagnosis, treatment, or prevention of diseases associated with cell proliferation and immune response. is there. Table 1 shows the SEQ ID No., Incyte clone number, cDNA library, NCB for each human / regulatory protein disclosed herein.
I shows the SEQ ID NO and the description in GenBank.

[0080]

[Table 1]

HRGP-1 (SEQ ID NO: 1) was initially identified using a computer search for amino acid sequence alignments in Insights clone No. 1331739 originating from the PANCNOT07 cDNA library. SEQ ID NO: 1 of consensus sequence
No. 3 is Insight's clone No. 1529406 (origin of PANCNOT04), 883517 (PANCNO
T05 origin), and 1331739, 1329209, 1329359, 1328354, 1329158, and 132845
It is derived from the extension and assembly of 1 (originating PANCNOT07).

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. HRGP-1 has a length of 419 amino acids, and P (1
Signature sequence of the zinc carboxypeptidase / zinc binding region spanning No. 70) -F (No. 203) and H (No. 306) -Y (No. 317)
One cAMP- and cGMP-dependent protein kinase phosphorylatable site at T (No. 197); S (No. 29), S (No. 31), T (No. 88), S (No. 95), T (No. 124), T (221), S (282), S (288), S (
363), T (399) and T (409) at 11 casein kinase II phosphorylatable sites; S (167), T (232), T (384),
And T (# 399) at four protein C phosphorylatable sites; and T (11
No. 9) has one tyrosine kinase phosphorylatable site. HRGP-1
Has sequence homology to human carboxypeptidase A (GI 35330). HRG
The mRNA encoding P-1 was expressed in a cDNA library derived from gastrointestinal tract tissue, especially pancreas, and has been implicated in cancer and diabetes.

HRGP-2 (SEQ ID NO: 2) was initially identified using a computer search for amino acid sequence alignments in Insights clone No. 1345619, originating from the PROSNOT11 cDNA library. SEQ ID NO: 1 of consensus sequence
4 are Insights clone Nos. 1345619 (originating from PROSNOT11), 2732826 (OVART
UT04), 1447240 (PLACNOT02 origin), 3598860 (DRGTNOT01 origin), 16
86916 (origin of PROSNOT15), 410406 (origin of EOSIHET02), and 345964 (origin of THYMNOT
02 originated from the extension and assembly.

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. HRGP-2 has a length of 403 amino acids and has a residue K
2 from (103) to F (110) and R (181) to M (188)
Two eukaryotic putative RNA binding region PNP-1 signature sequences; two glycosylable sites at N (46) and N (47); S (54), T (7
4) Casein Kinase II at S (No. 151) and T (No. 390)
Phosphorylation-possible sites; and S (No. 90), T (No. 99), S (No. 169), T (1
79 (SEQ ID NO: 79), T (191), and T (276) having six phosphorylatable sites of protein kinase C. HRGP-2 is a human RNA binding protein S
It has sequence homology to CR2 (GI 558529). MRNA encoding HRGP-2 was expressed in cDNA libraries derived from actively proliferating cells, particularly those associated with cancer or the immune response, and cells associated with reproductive and nervous system tissues.

HRGP-3 (SEQ ID NO: 3) was initially identified using a computer search of amino acid sequence alignments in Incyte Clone No. 1442636, originating from the THYRNOT03 cDNA library. SEQ ID NO: 1 of consensus sequence
No. 5 is Insight No. clone 1442636 (originating from THYRNOT03), 1548951 (RPOSN
OT06) (originated from OT06) and 930473 and 930805 (originated from CERVNOT01).

In one embodiment, the invention encompasses a polypeptide having the amino acid sequence of SEQ ID NO: 3. HRGP-3 has a length of 334 amino acids and an inorganic pyrophosphatase signature sequence spanning residues D (position 164) to V (position 170); 2 at N (position 54) and N (position 289). N-glycosylatable sites; S
(S72), T (148), S (179), T (303), S (309) and S (322) at the six phosphorylatable sites of casein kinase II; and S (No. 28) has one protein kinase C phosphorylatable site.
HRGP-3 has sequence homology to yeast inorganic pyrophosphatase (GI 4199). MRNA encoding HRGP-16 is expressed in cancer-related cDNA libraries (46%) and inflammation-related cDNA libraries (30%), especially cDNA libraries from genital, cardiovascular, and gastrointestinal tract tissues. Was.

HRGP-4 (SEQ ID NO: 4) was first identified in Incyte Clone No. 1458327 from the COLNFET02 cDNA library using a computer search for amino acid sequence alignment. SEQ ID NO: 1 of consensus sequence
No. 6 is Insight's clone No. 1458327 (origin of COLNFET02), 3224639 (UTRSN
OT03 origin), 022648 (origin of ADENINB01), 2185537 (origin of PROSNOT26), 546
947 (origin of BEPINOT02), 993339 (origin of COLNNOT11), 1615883 (origin of BRAITUT12), 1538280 (origin of SINTTUT01), and derived from the extension and assembly of 1419851 (origin of KIDNNOT09).

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 4. HRGP-4 has a length of 623 amino acids and has a residue F
Signature sequence of the ABC transporter family ranging from (229) to L (243); ATP / GTP binding site motif (P loop) ranging from residues G (430) to S (437); S (110) and Two amidable sites at I (131); four N-glycosylable sites at N (82), N (90), N (400), and N (516); CAMP- and cG at S (No. 458)
MP-dependent protein kinase phosphorylatable site; T (No. 51), T (104
No., T (No. 316), and four casein kinase II phosphorylation sites at S (No. 478); S (No. 110), S (No. 154), T (No. 167), T (No. 27)
No. 3), S (No. 349), T (No. 372), S (No. 377), S (No. 402),
T (# 506) and a protein kinase C phosphorylatable site at T (617); and a tyrosine kinase phosphorylatable site at Y (# 601).
HRGP-4 has sequence homology to members of the yeast ABC transporter protein family (GI 507374). MRNA encoding HRGP-4 may be a cDNA library derived from actively proliferating cells, particularly cancer or immune response related cDN.
It was expressed in the A library.

HRGP-5 (SEQ ID NO: 5) was initially identified in Incyte Clone No. 1686892 from the PROSNOT15 cDNA library using a computer search for amino acid sequence alignment. SEQ ID NO: 1 of consensus sequence
No. 7 is Insight No. 003036 (originating from HMC1NOT01), 754127 (BRAITUT
02 origin), 1235963 (origin from LUNGFET03), 1412956 (origin from BRAINOT12), 1645
848 (originating from PROSTUT09), 1688892 (originating from PROSNOT15), and 3215905 (originating TESTNOT
07 (origin).

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 5. HRGP-5 has a length of 437 amino acids and has residues G
(No. 120) ABCAGS-ABC binding site motif A (P loop); S (6
Casein kinase II phosphorylatable sites at positions No. 8), S (No. 77), T (No. 157), S (No. 185), S (No. 312), and T (No. 343); and S (No. 5)
, S (No. 142), T (No. 147), T (No. 157), S (No. 2007), T (No.
318) and S (432) at the protein kinase C phosphorylatable site. HRGP-5 has sequence homology to GTP-binding proteins originating from E. coli (GI 1033155). MRNA encoding HRGP-5 may be a cDNA library derived from actively proliferating cells, particularly cancer or immune response related cDN.
It was expressed in the A library.

HRGP-6 (SEQ ID NO: 6) was initially identified using a computer search for amino acid sequence alignments in Incyte Clone No. 1846116 from the COLNNOT09 cDNA library. SEQ ID NO: 1 of consensus sequence
8 are Insights clone No. 776000 (origin of COLNNOT05), 954544 (KIDNNOT
05 origin), 1846116 (origin from COLNNOT09), 1856648 (origin from PROSNOT18), and
It is derived from the extension and assembly of 2183017 (origin of SININOT01).

In one embodiment, the invention includes a polypeptide comprising the amino acid sequence of SEQ ID NO: 6. HRGP-6 has a length of 483 amino acids, and G (4
C-terminal amidable site at T (23); T (2), S (43), S (58)
No.), T (No. 95), S (No. 190), S (No. 276), T (No. 297), T (No.
Casein kinase II phosphorylatable sites at S (No. 301), S (No. 345), S (No. 350), and S (No. 351); S (No. 174), S (No. 232), S (27)
6), T (276), T (297), S (361), and S (372) at the protein kinase C phosphorylation site; and tyrosine kinase phosphorylation at Y (388) Has a possible site. HRGP-6 is a nematode cDNA yk8
It has sequence homology to members of 9e9.5 (GI 1213557). MRNA encoding HRGP-6 is expressed in a cancer-related cDNA library (54%), particularly a cDNA library for prostate, lung, colon, breast and brain tumors, and an immune response-related cDNA library ( 23%).

[0093] HRGP-7 (SEQ ID NO: 30) was initially identified using a computer search of amino acid sequence alignments in Incyte Clone No. 1913206 originating from the PROSTUT04 cDNA library. SEQ ID No. of the consensus sequence:
No. 19 is Insight No. 897272 (origin of BRSTNOT05), 917341 (BRSTN
OT04 origin), 1260595 (origin SYNORAT05), 1913206 (origin PROSTUT04), and 3224569 (origin UTRSNON03).

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 30. HRGP-7 has a length of 543 amino acids and is a signal peptide sequence generally ranging from residues M (No. 1) to I (No. 34); internal myristoylation within the signal peptide at G (No. 28) Site; N (No. 57), N (1
09), N (200), N (204), N (228), and N (534)
No.) at the N-glycosylation-possible site; S (No. 13), S (No. 97), S (186)
No.), S (No. 213), S (No. 254), S (No. 361), S (No. 387), S
(No. 428), and the casein kinase II phosphorylatable site at S (No. 538); and S (No. 4), S (No. 31), S (No. 90), S (No. 97), S (No. 186) ), S (No. 361), S (No. 420), and S (No. 538). HRGP-7 has sequence homology to porcine gastric mucin protein (GI 915208). MRNA encoding HRGP-7 is a cDNA library derived from actively growing cells, particularly cancer-related (42%),
It was expressed in an immune response related (32%) and fetal development related (18%) cDNA libraries.

[0095] HRGP-8 (SEQ ID NO: 8) was initially identified using a computer search for amino acid sequence alignments in Insights clone No. 2637177, originating from the BONTNOT01 cDNA library. SEQ ID NO: 2 of consensus sequence
0 is Insight Clone No. 2014984 (origin of TESTNOT03) and 2637177 (BON
TNOT01) and the extension and assembly of the shotgun sequences SAEA00455, SAEA00561, and SAEA01588.

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 8. HRGP-8 has a length of 180 amino acids and has N (5
7) and two N-glycosylation sites at N (124); glycosaminoglycan attachment sites at S (116): and T (5), Y (45),
S (No. 48), T (No. 76), T (No. 84), S (No. 135), and S (149)
No. 7). HRGP-8 is a nematode protein
It has sequence homology to C43E11.9 (GI 1703574).

HRGP-9 (SEQ ID NO: 56) was initially identified using a computer search of amino acid sequence alignments in Incyte Clone No. 3026841 originating from the HEARFET02 cDNA library. SEQ ID No. of the consensus sequence:
21 are Insights clone No. 3092189 (originating from HEARFET02), 2494035 (ADR
ETUT05 origin), 489738 (origin HNT2AGT01), 1493228 (origin PROSNON01), 2
106486 (origin of BRAITUT03), 2749492 (origin of BRSTTUT14), 2111992 (origin of BRAITUT0
3 originated from 1874754 (originated from LEUKNOT02), and 1513059 (originated from PANCTUT01).

[0098] In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 9. HRGP-9 has a length of 130 amino acids and N (1
Two N-glycosylatable sites at No. 4) and N (No. 59); and T (No. 16), S (No. 33), S (No. 47), S (No. 61), Y (No. 62) , S (No. 70)
, S (No. 90), S (No. 104), and S (No. 116). HRGP-9 has sequence homology to a human protein (GI 2196870) that is abundant in diabetes. MRNA encoding HRGP-9 is a cDNA library derived from actively growing cells, especially cancer-related cDNAs.
Expressed in library (50%).

HRGP-10 (SEQ ID NO: 10) was initially identified using a computer search for amino acid sequence alignments in Incyte Clone No. 3119737 originating from the LUNGTUT13 cDNA library. The consensus sequence SEQ ID NO: 22 corresponds to Incyte clone No. 3119737 (origin of LUNGTUT13), 1854190 (H
NT3AZT01 origin), 772126 (COLNCR01 origin), 1443080 (THYRNOT03 origin),
1453628 (origin of PENITUT01), and 1538342 (origin of SINTTUT01) derived from the extension and assembly.

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 10. HRGP-10 has a length of 193 amino acids and has a T
Three casein kinases at (No. 37), S (No. 73), and T (No. 127)
II phosphorylatable site; one AT spanning residues T (127) to S (160)
It has a P binding site motif (P loop); and one prenyl group binding site at residue C (number) (CAAX box). HRGP-10 has sequence homology to the human rhoC coding region (GI 36034). The results of Northern analysis indicate the expression of HRGP-10 in various libraries. At least 52% of such libraries are immortalized or cancerous and at least 30% are immune response related.

HRGP-11 (SEQ ID NO: 11) was initially identified using a computer search for amino acid sequence alignments in Incyte Clone No. 3257165 originating from the OVARTUN01 cDNA library. The consensus sequence SEQ ID NO: 23 is obtained from Incyte clone No. 3257165 (originating from OVARTUN01), 1976041 (P
Derived from the extension and assembly of ANCTUT02), 862467 (origin of BRAITUT03), and 1352543 (origin of LATRTUT02).

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 11. HRGP-11 has a length of 202 amino acids and has a T
(No. 94) at a cAMP- and cGMP-dependent protein kinase phosphorylatable site; S (187) at a casein kinase II phosphorylatable site; G (2
Two N-myristoylizable sites at No. 3) and G (No. 27); and S (31
No.), T (No. 43), T (No. 60), T (No. 71), S (No. 74), S (No. 89), T (No. 94), and Protein Kinase C at T (No. 97) It has a phosphorylatable site. HRGP-11 has sequence homology to rat PTTG (GI 1763265). The results of analysis by the Northern method indicate expression of HRGP-11 in various libraries. At least 48% of such libraries are immortalized or cancerous, at least 29% are immune response related, and at least 32% are fetal disease related.

HRGP-12 (SEQ ID NO: 12) was initially identified using a computer search for amino acid sequence alignments in Incyte clone No. 3371455 originating from the CONNTUT05 cDNA library. The consensus sequence SEQ ID NO: 24 corresponds to Incyte clone No. 3371455 (origin of CONNTUT), 2210345 (SIN
TFET03 origin), 915388, 196186, and 918434 (origin BRSTNOT04), 760643 (B
RAITUT02 origin), 674891 (origin CRBLNOT01), 3526393 (origin of ESOGTUN01), 968807 (origin of BRSTNOT05), 925515 (origin of BRAINOT04), 1997822 (origin of BRSTTUT)
03 origin), 2149413 (origin BRAINOT09), 1210219 (origin BRSTNOT02), and
Derived from the extension and assembly of 1939856 (origin of HIPONOT01).

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 12. HRGP-12 has a length of 387 amino acids, and S
(No. 152), a cAMP- and cGMP-dependent protein kinase phosphorylatable site; S (No. 10), S (No. 62), S (No. 64), S (No. 89), T (No.
No. 107), T (No. 145), S (No. 228), S (No. 230), T (No. 243), S (No. 269), S (No. 346), S (No. 356), and T (No. 367) No. 13) at the site capable of phosphorylating casein kinase II; T (No. 107), T (14)
5), four protein kinase C phosphorylatable sites at S (269) and T (314); one cell-adhesive sequence at residue R (100); and C (384). No.) It has one phenyl group binding site (CAAX box) in SIM. HRGP-12 has 100% sequence homology with human KIAA0270 (GI 1665807). From the results of Northern analysis, it was found that HRGP-
70 expression can be seen. At least 44% of such libraries are immortalized or cancerous and at least 21% are associated with fetal disease.

The invention also encompasses HRGP variants that retain the biological or functional activity of HRGP. Preferred variants of HRGP are those that have at least about 80%, more preferably at least about 90%, amino acid sequence identity with the amino acid sequence of HRGP. Most preferred HRGP variants are those that have at least 95% sequence identity with the HRGPs disclosed herein.

The present invention also encompasses a polynucleotide encoding HRGP. Therefore,
Any nucleic acid sequence encoding the amino acid sequence of HRGP can be used to create a recombinant molecule that expresses HRGP. In certain embodiments, the present invention relates to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 1.
7, a polynucleotide consisting of a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 Is included.

As will be appreciated by those skilled in the art, the degeneracy of the genetic code results in a wide variety of HRGPs, including those that have minimal homology to the nucleotide sequence of any known naturally occurring gene. Can be created. Thus, the present invention includes within its scope all possible nucleic acid sequence variations created by selecting combinations based on the possible codon choices. Their combination is
It is based on the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring HRGP, and all such variations are to be considered as specifically disclosed herein.

The nucleotide sequence encoding HRGP and its variant sequences are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring sequence under appropriately selected stringency conditions, but have substantially different codons. HRGP you have
Alternatively, it may be beneficial to create a nucleotide sequence that encodes a derivative thereof. In selecting codons, it can be chosen to increase the incidence of peptide expression in a particular prokaryotic or eukaryotic expression host, depending on the frequency with which particular codons are used by the host. Other reasons for substantially altering the nucleotide sequence encoding HRGP and its derivatives so that the encoded amino acid sequence remains unchanged, such as a longer half-life than transcripts made from naturally occurring sequences Creating RNA transcripts with properties. The scope of the present invention also includes the production of a DNA sequence encoding HRGP or a derivative thereof, or a fragment thereof, by completely synthetic chemistry. Once made, the synthetic gene can be inserted into various available expression vectors and cell lines using well-known reagents. In addition, synthetic chemistry can be used to introduce mutations into the HRGP-encoding sequence or any fragment thereof.

Also included within the scope of the present invention are various stringency conditions (
See, for example, the nucleotide sequences claimed under Wahl, GM and SLBerger (1987; Methods in Enzymol. 152: 399-407) and Kimmel, AR (1987; Methods in Enzymol. 152: 507-511). In particular, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 1
9, there are polynucleotide sequences that can hybridize with the nucleotide sequences shown in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.

[0110] DNA sequencing methods are well known and commonly available to those of skill in the art, and can be used to practice any of the embodiments of the present invention. In this method, for example, Sequenase which is a Klenow fragment of DNA polymerase I (US Biochemical
Corp. Cleveland OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago IL), or a calibration exo such as that found in the ELONGASE Amplification System available from Gibco BRL (Gaithersburg MD). Enzymes, such as a combination of a nuclease and a recombinant polymerase can be used. This process is based on the Hamilton Micro Lab2200 (Hamilton, Reno
, NV), Peltier Thermal Cycler (PTC200; MJ Reserch, Watertown MA) and
It is preferred to automate using equipment such as ABI Catalyst and ABI377 and 377 DNA sequencers (Perkin Elmer).

The nucleic acid sequence encoding HRGP can be extended using partial nucleotide sequences using various well-known methods to detect upstream sequences such as promoters and regulatory elements. For example, in a restriction site PCR method that is one of the methods that can be used, an unknown sequence adjacent to a known position is obtained using a universal primer (for example, Sarkar, G. (1993) PCR Methods Applic 2: 318-322). reference). Specifically, genomic DNA is first amplified in the presence of a primer specific to a known region and a primer to a linker sequence. PCR of the amplified sequence using the same linker primer and another specific primer contained within the first primer
Take the second round. The product of each round of PCR is transcribed using the appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR can also be used to amplify or extend sequences using various primers based on known regions (eg, Triglia, T. et al. (1988) Nucleic Aci.
ds Res 16: 8186). Primers were purchased using commercially available software such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth MN) or other suitable programs and were 22-30 nucleotides in length and 50% in GC content.
% And is designed to anneal to the target sequence at a temperature of about 68-72 ° C.
In this method, several restriction enzymes are used to generate appropriate fragments of a known region of the gene. This fragment is then circularized by intramolecular ligation and used as a template for PCR.

Another method that can be used is capture PCR, in which DNA fragments adjacent to known sequences in human and yeast artificial chromosomal DNA are amplified by PCR (eg, Lagerstrom, M. et al. (1991). ) PCR Methods Applic 1: 111-119)
. In this method, the recombinant double-stranded sequence can also be incorporated into an unknown fragment of the DNA molecule by digestion and ligation with a plurality of restriction enzymes before performing PCR.

Another method that can be used to obtain unknown sequences is Parker, JD et al. (See, eg, 1991; Nucleic Acids Res 19: 3055-3060). Furthermore, PC
R, nested primers, genomic DNA using PromoterFinder ^™ library
You can walk inside (Clontech, Palo Alto CA). This process is useful for finding intron / exon junctions without having to screen the library.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Random primed libraries are also preferred in that they contain more sequences containing the 5 'region of the gene. The use of a randomly primed library is particularly preferable when an oligo d (T) library cannot obtain a full-length cDNA. Genomic libraries can also be useful for extending sequence to a non-transcribed 5 'regulatory region.

A commercially available capillary electrophoresis system can be used to size-analyze the nucleotide sequence of a product of sequencing or PCR and confirm its presence. In particular, capillary sequencing uses a flowable polymer for electrophoretic separation, four different laser-activated fluorescent dyes (one for each nucleotide), and a wavelength emitted by a CCD camera. Is detected. The output / light intensity is converted to electrical signals using appropriate software (eg, Genotyper ^™ and Sequence Navigator ^{™ from} Perkin Elmer), and the entire process from sample loading to computer analysis and electronic data display is computer controlled. You. Capillary electrophoresis is a method of DNA that is present in small amounts in a particular sample.
Particularly suitable for the sequencing of small pieces of

In another embodiment of the invention, a polynucleotide sequence encoding HRGP, or a fragment thereof, is incorporated into a recombinant DNA molecule to provide a recombinant DNA molecule with an HRP in a suitable host cell.
Expression of RGP, a fragment thereof, or a functional equivalent can be induced. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially identical, ie, functionally equivalent, amino acid sequences can be created, and these sequences can be used for HRGP cloning and expression. it can.

As will be appreciated by those skilled in the art, it may be beneficial to create a HRGP coding nucleotide sequence with non-naturally occurring codons. For example, selecting codons that are preferred in a particular prokaryotic or eukaryotic host to increase protein expression or have desirable properties such as a longer half-life than transcripts generated from naturally occurring sequences. Can be created.

[0119] The nucleotide sequences of the present invention can be recombined using well-known methods to modify the HRGP-encoding sequence for various purposes. The purpose of this sequence modification includes, but is not limited to, for example, cloning, processing and / or altering expression of the gene product. DNA rearrangement by random fragments and PCR-based reassembly of gene fragments and synthetic oligonucleotides (reassembly
) Allows the nucleotide sequence to be recombined. For example, site-directed mutagenesis can result in the insertion of new restriction sites, altered glycosylation patterns, altered codon preferences, generation of splice variants, and introduction of mutations.

[0120] In another embodiment of the invention, the original HRGP coding sequence, the modified HRGP
The coding sequence, or the recombinant HRGP coding sequence, can be ligated to a heterologous sequence to provide a sequence encoding a fusion protein. For example, HRGP
To screen for inhibitors of activity from a peptide library, it may be useful to encode a chimeric HRGP protein that is recognized by commercially available antibodies. The fusion protein can also be designed to have a cleavage site at a position between the HRGP-encoding sequence and the heterologous protein sequence, which results in a HRGP.
The GP can be cleaved and purified separately from the heterologous portion.

In another embodiment of the present invention, known chemical methods (eg, Caruthers. MH et al. (198)
0) Nucl. Acids Res. Symp. Ser. 7: 215-223; Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 225-232) can be used to synthesize the entire HRGP-encoding sequence or a portion thereof. Alternatively, the protein itself can be produced using chemical methods to synthesize the HRGP amino acid sequence or a fragment thereof. For example, various solid-phase techniques (eg, Roberge, JY et al. (1995) Science 26
9: 202-204), and the synthesis can be automated, for example, by ABI
This can be achieved by using a 431A peptide synthesizer (Perkin Elmer).

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (eg, Chiez. RM and FZ Regnier (1990).
Methods Enzymol. 182: 392-421). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (eg, Creighton
T. (1983) Proteins Structure and Molecular Principles , WH Freeman and Co
., NY). Further, by modifying the amino acid sequence of HRGP or any part thereof during its direct synthesis, and / or by using a chemical method to bind to a sequence derived from another protein or any part thereof, , Mutant polypeptides can be created.

In order to express biologically active HRGP, the nucleotide sequence encoding HRGP or a functional equivalent thereof is converted to an appropriate expression vector, ie, the elements necessary for transcription and translation of the inserted coding sequence. Insert into a vector containing

[0124] Methods known to those of skill in the art can be used to construct expression vectors containing a sequence encoding HRGP and appropriate transcriptional and translational control regions. These methods include in vitro recombinant DNA technology, synthetic technology, and in vivo gene recombination technology (eg, Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press). , Planview NY and Ausubel, FM other Cu rrent Protocol in Molecular Biology, John Wiley & Sons, New York, see NY).

A variety of expression vector / host systems can be used to maintain and express HRGP-encoding sequences. Such things include, but are not limited to, recombinant bacteriophages, microorganisms such as bacteria transformed with plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors,
Insect cell lines infected with viral expression vectors (eg baculovirus),
Plant cell lines or animal cell lines transformed with a virus expression vector (for example, cauliflower mosaic virus CaMV, tobacco mosaic virus TMV) or a bacterial expression vector (for example, Ti or pBR322 plasmid) can be mentioned. The invention is not limited by the host cell used.

“Regulatory regions” or “control sequences” are the untranslated regions of a vector that interact with host cell proteins for transcription and translation, ie, enhancers, promoters, and 3 ′ untranslated regions. The strength and specificity of the action of such elements can vary. Depending on the vector system and host used, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning into bacterial systems, Bluescript phagemid (Stratagene, LaJolla CA) or pSportI ^™ plasmid (Gibco BRL
) Can be used, such as a hybrid lacZ promoter. In insect cells, the baculovirus polyhedrin promoter can be used. A promoter or enhancer derived from the genome of a plant cell (
For example, a heat shock RUBISCO and storage protein gene), or a promoter or enhancer from a plant virus (eg, a viral promoter or leader sequence) may be cloned into the vector. For mammalian cell lines, promoters from mammalian genes or from mammalian viruses are suitable.
If it is necessary to generate a cell line that contains multiple copies of the HRGP-encoding sequence, vectors based on SV40 or EBV can be used with appropriate selectable markers.

In bacterial systems, a wide variety of expression vectors may be selected depending on the use of the HRGP. For example, when a large amount of HRGP is required for antibody induction, a vector that can express a fusion protein that can be easily purified at a high level can be used. Such vectors include, but are not limited to, a multifunctional E. coli cloning and expression vector, Bluescript (Stratagene) (in which the sequence encoding HRGP is replaced with an amino-terminal methionine and a subsequent β-galactosidase). And a pIN vector (Van Heeke, G. and S. et al.) That can bind to the frame of a vector having a sequence of seven residues of
M. Schuster (1989) J. Biol. Chem. 264: 5503-5509). In addition, a pGEX vector (Promage, Madison WI) can be used to express a foreign polypeptide as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption to glutathione agarose beads followed by elution in the presence of free glutathione. The protein produced in such a system can be designed to include a heparin, thrombin or factor Xa protease cleavage site, allowing the cloned polypeptide of interest to be released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae , a wide variety of vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase and PGH can be used (eg, Ausubel et al., Supra) and G
rant et al. (1987) Methods Enzymol 153: 516-544).

When using plant expression vectors, expression of the HRGP-encoding sequence can be driven by any of a number of promoters. Use of viral promoters, such as the CaMV 35S and 19S promoters, alone or in combination with an omega leader sequence from TMV (see, eg, Takamatsu, N. et al. (1987) EMBO J 6: 307-311). Can be. Alternatively, plant promoters such as the small subunit of RUBISCO, the heat shock promoter, may be used (eg, Coruzz
i, G. et al. (1984) EMBO J 3: 1671-1680); Broglie, R. et al. (1984) Science 224: 83.
8-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105). These products can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in various publicly available documents (eg, Hobbs, S. or Murry, L. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill NY, pp 191-196).
See .E.).

Insect systems can also be used for expression of HRGP. For example, such a system
In one of the yotoga (Spodoptera frugiperda) Cells or nettleTrichoplusia) As a vector for expressing foreign genes in larvae, Autographa californicaUse nuclear polyhedrosis virus (AcNPV). HRGP coding sequences are non-essential for viruses such as the polyhedrin gene.
Can be cloned into a region and placed under the control of the polyhedrin promoter
. Successful insertion of the HRGP coding sequence inactivates the polyhedrin gene.
A mutant virus lacking the coat protein membrane is generated. Next, this mutant
Ils, Yotouga (S.frugiperda) Cells or nettle (Trichopl usia ) Infect larvae in which HRGP can be expressed (Engelhar
d, E.K. et al. (1994) Proc. Nat. Acad. Sci. 91: 3224-3227).

[0131] In mammalian host cells, a variety of viral-based expression systems may be utilized. When an adenovirus is used as an expression vector, HRG
The sequence encoding P can bind into an adenovirus transcription / translation complex consisting of the late promoter and tripartite leader sequence. Non-essential E1 or E3 in the viral genome
Insertion into the region yields a live virus capable of expressing HRGP in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:36).
55-3659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells.

By using human artificial chromosomes (HACs), it is possible to supply a DNA fragment larger than a DNA fragment that can be expressed in a plasmid. For therapeutic purposes, 6-10 M HACs can be constructed and supplied using conventional delivery methods (liposomes, polycationic amino polymers, or vesicles).

[0133] Certain initiation signals are also required for more efficient translation of the sequence encoding HRGP. Such signals include the ATG initiation codon and adjacent sequences. If HRGP and its initiation codon and upstream sequences are inserted into the appropriate expression vector, no other transcriptional or translational control signals are required. However, if only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal, including the ATG initiation codon, must be provided. In addition, the start codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be from a variety of sources, both natural and synthetic. For example, literature (eg, Scharf
, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162) by including appropriate enhancers in the particular cell line used.
The efficiency of expression can be increased.

In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences, or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation (lipida
tion) and acylation. Post-translational processing that cuts off the "prepro" portion of the protein is also important for correct insertion, folding, and / or performance. A variety of host cells (eg, CHO, HeLa, MDCK, HEK293, and WI3) having specific cellular devices and characteristic mechanisms for such post-translational effects.
8) is available from the American Type Culture Collection (ATCC; Bethesda, MD) and can be selected from among them to ensure correct modification and processing of the foreign protein to be introduced.

[0135] Stable expression is desirable to ensure high yield production of the recombinant protein over an extended period of time. For example, a cell line that stably expresses HRGP is transformed by using an expression vector containing the viral origin of replication and / or an endogenous expression element and a selectable marker gene on the same vector or on another vector. be able to. After the introduction of the vector, the cells are allowed to grow for 1-2 days in a concentrated medium before switching to a selective medium. The purpose of the selectable marker is to confer resistance for selection and allow cells expressing the introduced sequence to grow and recover based on its presence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.

[0136] Any number of selection systems can be used to recover transformed cell lines. Selection systems include, but are not limited to, herpes simplex virus thymidine kinase (tk) (see, eg, Wigler, M. et al. (1977) Cell 11: 223-32) and adenine phosphoribosyltransferase (aprt) ( For example, Lowy, I. et al. (19
80) Cell 22: 817-823 reference) have genes like, respectively tk ^- used Te cells odor ^- or aprt. Also, resistance to antimetabolites, antibiotics or herbicides can be used as a basis for selection. For example, dhfr confers resistance to methotrexate (see, eg, Wigler, M. et al. (1980) Proc. Natl. Acad. Sci. 77: 3567), and npt confers resistance to the aminoglycosides neomycin and G-418 ( For example, Colberre-
Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14), als or pat confers resistance to chlorsulfuron, phosphinotricin acetyltransferase (Murry, ed. Out). Other genes available for selection include trpB, which allows cells to use indole instead of tryptophan, and hisD, which allows cells to use histinol instead of histidine ( For example, Hartman, SC and RC Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047.
-51). More recently, not only to identify transformants, but also to quantify the amount of transient or stable protein expression by a particular vector system,
For example, visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin have been used (for example, Rhodes, CA et al. (1995) Methods Mol. Biol. 55). : 121-
131).

Although the presence / absence of marker gene expression also indicates the presence of the gene of interest,
It may be necessary to confirm its presence and expression. For example, when a sequence encoding HRGP is inserted into a marker gene sequence, a transformed cell containing a sequence encoding HRGP can be confirmed by lacking the function of the marker gene. Alternatively, a marker gene can be placed in tandem with the sequence encoding HRGP, both under the control of a single promoter. Expression of the marker gene in response to induction or selection will usually also indicate expression of the tandemly arranged sequence.

Alternatively, host cells that contain the HRGP-encoding nucleic acid sequence and express HRGP can be identified by various methods well known to those skilled in the art. Such methods include, but are not limited to, DNA-DNA or DNA-RNA hybridization, and techniques using membranes, solutions, or chips for detecting and / or quantifying nucleic acids and proteins. DNA using a probe, part or fragment of a sequence encoding HRGP, such as protein bioassay or immunoassay.
HRGP by DNA or DNA-RNA hybridization or amplification
Can be detected. In assays utilizing nucleic acid amplification, oligonucleotides or oligomers based on HRGP-encoding sequences are used to detect transformants containing HRGP-encoding DNA or RNA.

Various protocols are well known for detecting and measuring HRGP expression, using either polyclonal or monoclonal antibodies specific for this protein. Examples of such protocols include enzyme linked immunoassays (ELISA).
), Radioimmunoassay (RIA) and Fluorescent Display Cell Sorter (FACS)
Is mentioned. A two-site, monoclonal-based immunoassay utilizing a monoclonal antibody reactive to two non-interfering epitopes on the HRGP polypeptide is preferred, but a competitive binding assay may also be used. Can be. These and other assays have been described elsewhere (see, for example, Hampton, R. et al. (1990) Serological Methods, a Laboratory Manual , APS Press, St. Paul MN and Maddox, DE et al. (1983). ) J. Exp.
Med. 158: 1211-1216).

In addition, various labeling and conjugation techniques are well known and can be used in various nucleic acid and amino acid assays. As a means for producing a labeled hybridization probe or PCR probe for detecting closely related sequences,
Examples include oligo labeling, nick translation, terminal labeling, and PCR amplification using labeled nucleotides. Alternatively, the sequence encoding HRGP or any fragment thereof is cloned into a vector for the production of an mRNA probe. Such vectors are well known and commercially available, and can be used to synthesize RNA probes in vitro by adding an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. it can. Such methods are described in various commercially available kits (Pharmacia & Upjohn (Kalamazoo, MI); Promega
(Madison WI); and US Biochemical Corp. (Cleveland OH)). A suitable reporter molecule used to facilitate detection,
That is, examples of the label include a radionuclide, an enzyme, a fluorescein (fluorescent agent), a chemiluminescent agent or a coloring agent, a substrate, a cofactor, an inhibitor, and a magnetic particle.

[0141] Host cells transformed with the nucleotide sequence encoding HRGP can be cultured under conditions suitable for expressing the protein in the cell culture medium and recovering it therefrom. Proteins produced by the transformed cells are either secreted or included intracellularly, depending on the sequence and / or the vector used. As will be appreciated by those skilled in the art, expression vectors containing a polynucleotide encoding HRGP can be designed to include a signal sequence that directs secretion of HRGP across the cell membrane of a prokaryotic or eukaryotic cell. Still other constructs can be used to link a HRGP-encoding sequence to a nucleotide sequence encoding a polypeptide domain that facilitates purification of soluble proteins. Domains that facilitate such purification include, but are not limited to, metal chelating peptides such as the histidine tryptophan module, which allow purification on immobilized metals, and purification on immobilized immunoglobulins Protein A domain and FLAGS extension / affinity purification system (Immunex Corp., Seattle)
WA) and the like. Between the purification domain and the HRGP, for example, a factor Xa or enterokinase specific sequence (Invitrogen, Sa
Purification can be facilitated by the inclusion of a cleavable linker sequence such as (N Diego CA). One such expression vector expresses a nucleic acid sequence encoding six histidine residues, along with a sequence encoding HRGP, followed by a sequence encoding thioredoxin and an enterokinase cleavage site to create a fusion protein. . Histidine residues facilitate purification by immobilized metal ion affinity chromatography (IMIAC) and enterokinase cleavage sites provide a means for purifying HRGP from fusion proteins (eg, Porath
(1992) Prot. Exp. Purif. 3: 263-281 and Kroll, DJ et al. (1993) DNA C
ell Biol. 12: 441-453).

HRGP fragments can be produced not only by recombinant production methods but also by direct peptide synthesis using solid phase technology (eg, Merrifield
J. (1963) J. Am. Chem. Soc. 85: 2149-2154). Protein synthesis can be done manually, but can also be automated. Automated synthesis is described, for example, in Applied Bios
This can be performed using a ystem 431A peptide synthesizer (Perkin Elmer).
It is also possible to chemically synthesize the various HRGP fragments individually and combine them in a chemical manner to create the full-length molecule.

Therapeutic There is chemical and structural homology between the human and regulatory proteins of the present invention. Also, HRGP expression is strongly associated with cell proliferation. Therefore, it is desirable that HRGP is an activator, transcriptional regulator, or enhancer, and that expression of HRGP be reduced in cancers or immune responses that promote cell proliferation. When HRGP is an inhibitor or a suppressor and regulates or reduces cell proliferation, it is desirable to supply the protein or increase HRGP expression.

In one embodiment, when HRGP is an inhibitor, for the treatment or prevention of HRGP or a HRGP thereof, such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma. Fragments or derivatives may be administered to the patient. Such cancers include, but are not limited to, adrenal gland, bladder, bone, bone marrow, brain, breast, cartilage, cervix, gallbladder, ganglion, gastrointestinal tract, heart, kidney, liver, lung, muscle , Ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterine cancer.

In another embodiment, a pharmaceutical composition comprising purified HRGP can be used for the treatment or prevention of cancer, including but not limited to those listed above.

In another embodiment, an agonist specific for HRGP may be administered to a patient for the treatment or prevention of a cancer, including but not limited to those listed above.

In another embodiment, a vector capable of expressing HRGP or a fragment or derivative thereof can be administered to a patient for the treatment or prevention of cancer, including but not limited to those listed above.

In another embodiment, where HRGP promotes cell proliferation, in adenocarcinoma, leukemia,
For the treatment or prevention of cancer such as lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, an antagonist that decreases HRGP expression or activity may be administered to the patient for HRGP expression or treatment. Such cancers include, but are not limited to, adrenal gland, bladder, bone, bone marrow, brain, breast, cartilage, cervix, gallbladder, ganglion, gastrointestinal tract, heart, kidney, liver, lung, muscle , Ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterine cancer. In some embodiments, an antibody that specifically binds to HRGP may be used directly as an antagonist or jointly as a targeting or delivery mechanism to deliver a drug to cells or tissues that express HRGP. it can.

In another embodiment, a vector expressing a molecule complementary to a polynucleotide encoding HRGP is administered to a patient for the treatment or prevention of cancer, including but not limited to those listed above, in a patient. Can be administered.

Where HRGP promotes leukocyte activity or proliferation, an antagonist that reduces the activity of HRGP may be administered to the patient to treat or prevent an immune response. Such responses include, for example, AIDS, Addison's disease, adult respiratory distress syndrome, allergy, anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis, Dermatomyositis, diabetes, emphysema, atrophic gastritis,
Nephritis, gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocarditis or pericarditis, osteoarthritis, osteoporosis, pancreatitis, multiple Myositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, and autoimmune thyroiditis; complications of cancer and hemodialysis and extracorporeal circulation; viral, bacterial, fungal, parasite, parasite, and helminth infections And illnesses such as trauma.
In some embodiments, an antibody that specifically binds to HRGP may be used directly as an antagonist or jointly as a targeting or delivery mechanism to deliver a drug to cells or tissues that express HRGP. it can.

In another embodiment, to treat or prevent an immune response, including but not limited to those listed above, a vector capable of expressing a molecule complementary to a polynucleotide encoding HRGP is provided. It can be administered to a patient.

In another embodiment, HRGP or a fragment or derivative thereof can be provided to a cell to stimulate cell proliferation. Specifically, for the purpose of promoting cell growth and regeneration of tissues or organs, HRGP is prepared by in vitro delivery of cells or cells in culture using a liposome, virus-based vector, or a delivery mechanism such as electroinjection. It can be given to a group of cells. In particular, HRGP can be added to a cell, cell line, tissue or organ culture to stimulate cell proliferation for use in xenogeneic or autologous transplantation. In some cases, its ability to fight infection or cancer, or to correct a genetic defect in a disease such as, for example, sickle cell anemia, beta Mediterranean anemia, cystic fibromatosis, or Huntington's chorea about,
Select cells in advance.

In another embodiment, HRGP-specific agonists can be provided to the cells to stimulate cell proliferation as described above.

In another embodiment, cells may be provided with HRGP or a fragment thereof or an expressible vector to stimulate cell proliferation as described above.

In another embodiment, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention can be administered in combination with other suitable agents. One of skill in the art would be able to select an appropriate drug for use in combination therapy based on conventional pharmaceutical principles. By combining therapeutic agents, a synergistic effect that is effective in treating or preventing the various diseases described above can be obtained. By using this method, lower doses of each drug can increase the therapeutic effect and reduce the likelihood of side effects.

HRGP antagonists can be produced using well-known methods. Specifically, purified HRGP can be used to generate antibodies or to screen libraries of drugs to identify those that specifically bind to HRGP.

Antibodies to HRGP can also be generated using well-known methods. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments made from Fab expression libraries. Neutralizing antibodies (ie, those that inhibit dimer formation) are particularly suitable for therapeutic use.

A variety of hosts, such as goats, rabbits, rats, mice, etc., can be immunized by injecting HRGP or fragments or oligopeptides thereof having immunological properties to generate antibodies. Various adjuvants, depending on the species of the host, can be used to enhance the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, inorganic gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oily emulsions, Lulimpet hemocyanin, dinitrophenol, and the like. Among the adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are particularly preferred.

The oligopeptide, peptide, or fragment thereof used to elicit antibodies against HRGP has an amino acid sequence preferably consisting of 5 or more amino acids, more preferably 10 or more amino acids. Also, these sequences are preferably identical to a portion of the amino acid sequence of the original protein, and include the entire amino acid sequence of the small, naturally occurring molecule. Short stretches of HRGP amino acids can be fused with stretches of other proteins, such as keyhole limpet hemocyanin, to produce antibodies against the chimeric molecule.

The HRGP monoclonal antibody can be used in a continuous cell line in culture.
cell line) using a technique for producing antibody molecules. Such techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, EBV-hybridoma technology, and the like (eg, Kohler,
G. et al. (1975) Nature 256: 495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81
Cote, RJ et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; Cole, S .;
P. et al. (1984) Mol. Cell Biol. 62: 109-120).

In addition, "chimeric antibodies", such as techniques for splicing mouse antibody genes to human antibody genes, to obtain molecules with appropriate antigen specificity and biological activity.
Techniques developed for the production of (eg, Morrison, SL et al. (1984) Proc. Natl. Acad. Sci. 81: 6851-6855; Neuberger, MS et al. (1984).
Nature 312: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454). Alternatively, well-known techniques for the production of single-chain antibodies can be applied to create single-chain antibodies specific for HRGP by well-known methods. Antibodies with related specificities but differing idiotypic configurations can be created by chain shuffling from random combinatorial immunoglobulin libraries (eg, Burton DR (1991) Proc. Natl. Acad. Sci. 88: 11120-3).

In addition, literature (eg, Orlandi, R. et al. (1989), Proc. Natl. Acad. Sci. 86: 383)
3-3837; by screening a panel of highly specific binding reagents or a recombinant immunoglobulin library, as disclosed in Winter, G. et al. (1991) Nature 349: 293-299). Alternatively, antibodies can be produced by inducing production in lymphocyte populations in vivo.

Antibody fragments which contain specific binding sites for HRGP can also be generated. Such fragments include, but are not limited to, reducing F (ab ') ₂ fragments and F (ab') ₂ fragments that can be generated by digestion of antibody molecules with pepsin, Fab that can be created by
Fragments and the like. Alternatively, a monoclonal F having the desired specificity
Fab expression libraries may be generated so that ab fragments can be identified quickly and easily (see, eg, Huse, WD et al. (1989) Science 256: 1275-1281).

[0164] A variety of immunoassays can be utilized for screening to identify antibodies with the desired specificity. Various protocols for competitive binding assays or radioimmunoassays using either monoclonal or polyclonal antibodies with established specificity are well known in the art. In such an immunoassay, the amount of a complex formed between HRGP and its specific antibody is measured.
Two sites monoclonal based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes
oassay) is preferred, but a competitive binding assay may be used (Maddox, supra).

In another embodiment of the invention, a polynucleotide encoding HRGP, or any fragment or complement thereof, can be used for therapeutic purposes. In some embodiments, in situations where it is desirable to inhibit the transcription of mRNA, HRGP
The complementary sequence to the polynucleotide encoding can be used. Specifically, cells can be transformed with a sequence complementary to the polynucleotide encoding HRGP. Thus, complementary molecules or fragments can be used to modulate the activity of HRGP, ie to regulate gene function. Such techniques are now well known, and sense or antisense oligonucleotides, or larger fragments, can be designed from various locations in the coding and regulatory regions of the HRGP coding sequence.

Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia viruses, or expression vectors derived from various bacterial plasmids, are used for delivery of nucleotide sequences to target organs, tissues, or cells. be able to. Vectors expressing a nucleic acid sequence complementary to the polynucleotide of the gene encoding HRGP can be prepared using methods well known to those skilled in the art. These techniques are described in the literature (see Sambrook et al., Supra and Ausubel et al., Supra).

Genes encoding HRGP can be disrupted by transforming cells or tissues with an expression vector that expresses HRGP-encoding polynucleotides or fragments thereof at high levels. Using such a construct, a non-translatable sense or antisense sequence can be introduced into cells. Such vectors, even when not incorporated into DNA, continue to transcribe the RNA molecule until the vector is destroyed by an endogenous nuclease. Such transient expression can last for more than one month, even for non-replicating vectors, and even longer if appropriate replication elements are part of the vector system.

As described above, a sequence complementary to the control region, 5 ′ region, or regulatory region (signal sequence, promoter, enhancer, and intron) of the gene encoding HRGP, that is, an antisense molecule (DNA, RNA or By designing PNA), the way of gene expression can be changed. Oligonucleotides derived from the transcription initiation site, e.g., a region approximately between +10 and -10 of the start site, are preferred. Similarly, inhibition can be achieved using triple helix base pairing. Triple helix pairing is useful because it inhibits the ability of the double helix to unwind sufficiently for binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triple-helical DNA have been described in the literature (eg, Huber, BE and BI Carr, Molecular and Immunologic Protocols , Futura Publishing Co, Mt Kisco NY at Gee, JE et al. 1994)).
In order to inhibit the transcription of mRNA by preventing the binding of the transcript to the ribosome,
Complementary sequences, ie, antisense molecules, can also be designed.

[0169] Ribozymes, which are enzymatic RNA molecules, can also be used to catalyze the specific cleavage of RNA. In the mechanism of ribozyme action, a ribozyme molecule hybridizes to a complementary target RNA in a sequence-specific manner, followed by endonucleolytic cleavage. Examples of ribozymes that can be used include:
There are artificially synthesized hammerhead ribozyme molecules that can specifically and effectively catalyze the cleavage of HRGP-encoding sequences by endonucleases.

[0170] Specific ribozyme cleavage sites within the RNA that can be targeted are identified by first scanning the ribozyme cleavage sites containing the sequences GUA, GUU and GUC in the target molecule. Once identified, it is possible to evaluate the characteristics of the secondary structure that stops the function of the oligonucleotide for a short RNA sequence consisting of 15 to 20 ribonucleotides corresponding to the region of the target gene including the cleavage site. . The suitability of the candidate target is also determined by the ribonuclease protection assay (ribonucl
This can be evaluated by measuring the accessibility for hybridization with a complementary oligonucleotide by an ease protection assay.

[0171] Complementary ribonucleic acid molecules and ribozymes of the invention can be made by well-known methods for the synthesis of nucleic acid molecules. These techniques include oligonucleotide chemical synthesis techniques such as solid phase phosphoramidite chemical synthesis. Or,
RNA molecules can be created by the transcription of a DNA sequence encoding HRGP in vivo and in vitro. Such a DNA sequence can be incorporated into various vectors having a suitable RNA polymerase promoter, such as T7 or SP6. Alternatively, a cDNA construct that synthesizes constitutive RNA can be introduced into a cell line, cell or tissue.

[0172] RNA molecules can be modified to increase their stability in cells or to increase their half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'end and / or 3' end of the molecule, and phosphorothioate (rather than phosphodiesterase linkages) within the backbone of the molecule. phosph
orothioate) or 2'O-methyl. This method (
concept) is originally used in the production of PNAs and can be extended to all of these molecules as follows. That is, inosine, queosine, as well as by modification of acetyl-, methyl-, thio-, and similar forms of adenine, guanine, cytidine, thymine, and uridine that are not readily recognized by endogenous endonucleases. And the inclusion of less commonly used bases such as wybutosine, the system can be extended to all of these molecules.

Many methods are available for introducing vectors into cells or tissues,
The methods are equally suitable for in vivo , in vitro and ex vivo uses. In the case of ex vivo treatment, there is a method in which a vector is introduced into stem cells collected from a patient, propagated as a clone for autologous transplantation, and returned to the same patient. Delivery by transfection, liposome injection, or delivery by polycation amino polymer can be performed using methods well known in the art (for example, see Goldman, CK et al. (1997) Nature Biotechnology 15: 462-66).
.

Any of the above treatments can be applied to any suitable subject, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably humans.

In yet another embodiment of the present invention, a pharmaceutical composition is administered with a pharmaceutically acceptable carrier to achieve the above-mentioned therapeutic effects. Such a pharmaceutical composition comprises HRG
P, antibodies to HRGP, HRGP mimetics, agonists, antagonists, or inhibitors. This pharmaceutical composition comprises:
It is administered alone or together with one or more other drugs, such as, for example, stabilizers, using a sterile, biocompatible pharmaceutical carrier. Such carriers include, but are not limited to, saline, buffered saline, glucose or water. Such compositions can be administered to the patient alone or in combination with other drugs or hormones.

The route of administration of the pharmaceutical composition used in the present invention is not limited to the following, but may be oral administration, intravenous administration, intramuscular administration, intraarterial administration, intramedullary administration, intrathecal administration, Examples include intraventricular administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intranasal administration, enteral administration, topical administration, sublingual administration, and rectal administration.

[0177] These pharmaceutical compositions, in addition to the principal component, may contain suitable pharmaceutically-acceptable agents, such as excipients and additives, which facilitate the processing of the active ingredient into pharmaceutically usable formulations. Carrier. Details of the formulation or administration techniques can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton PA).

Pharmaceutical compositions for oral administration can be formulated in suitable dosage forms using pharmaceutically acceptable carriers well known in the art. With such a carrier, the pharmaceutical composition can be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for patients to take. it can.

Pharmaceutical preparations for oral administration are obtained by combining the main component and solid excipients and, if desired, grinding the resulting mixture to produce tablets or dragee cores. It is obtained by processing the mixture of granules after adding suitable additives if necessary. Suitable additives include sugar or protein fillers such as sugars including lactose, sucrose, mannitol or sorbitol, starches such as corn, wheat, rice, potatoes, methylcellulose, hydroxypropylmethylcellulose or carboxyl. There are celluloses such as sodium methylcellulose, gums such as gum arabic or tragacanth, and proteins such as gelatin or collagen. If desired, disintegrating or solubilizing agents may be added, or the cross-linked polyvinyl pyrrolidone, agar, alginic acid such as sodium alginate, or a salt thereof such as sodium alginate.

The dragee core is used by coating an appropriate tablet skin with a concentrated sugar solution or the like. Solutions for making the tablets include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or tablets for tablet identification or to indicate the amount or dosage of the principal component.

Orally administrable formulations include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a tablet, such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient in admixture with excipients or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, if desired, stabilizers. In soft capsules, the principal component is dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, liquid polyethylene glycol, with or without stabilizers.

Pharmaceutical preparations for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, and synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aminopolymers of non-lipid polycations can also be used for delivery. Appropriate agents or stabilizers may be added to the suspension, as desired, to increase solubility and allow for the preparation of highly concentrated solutions.

For topical or nasal mucosal administration, formulations are appropriate with penetrants appropriate to the particular barrier to be permeated. Such penetrants are well-known in the art.

The pharmaceutical compositions of the present invention can be prepared by any of the methods well known in the art, for example, mixing, dissolving, granulating,
Sugar coating, levigating, emulsifying, encapsulating, entrapping,
Alternatively, it is produced by freeze-drying.

The pharmaceutical composition can be provided as a salt, and can be formed with various acids, including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be more soluble in aqueous or protonic solvents than the corresponding free base forms. In addition, preferred formulations include those in the range of 4.5-5.5, buffered before use, from 1 mM to 5 mM.
There are lyophilized powders that contain all or any of 0 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol.

After the pharmaceutical composition has been prepared, it can be placed in an appropriate container and labeled for use in treating the indicated conditions. For administration of HRGP, such a label would indicate the amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the present invention are those that contain the active ingredient in an effective amount to achieve the desired purpose. Determination of an effective amount can be made well within the ability of those skilled in the art.

For any compound, the therapeutically effective amount can be estimated initially from assays of neoplastic cells or cell culture, usually in any animal model such as mouse, rabbit, dog, pig. This animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine effective doses and routes for administration in humans.

[0189] A therapeutically effective amount is an amount of an active ingredient that ameliorates a symptom or condition, for example, HRGP or a fragment thereof, an antibody to HRGP, an agonist, antagonist, or inhibitor of HRGP. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell culture media or using laboratory animals, eg, ED5
It can be determined as 0 (therapeutically effective amount in 50% of the population, 50% effective amount) and LD50 (50% lethal dose in the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50.

Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that achieve the ED50 with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, the sensitivity of the patient, as well as the route of administration.

The exact dosage will be selected by the attending physician, in light of factors related to the patient that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient and to maintain the desired effect. Factors to consider include the severity of the disease state, the patient's general health, the patient's age, weight, and gender, diet, time and frequency of administration, concomitant medications, response sensitivity, and resistance / response to treatment. And the like. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the clearance rate of the particular formulation.

Typical dosages are in the range of 0.1-100,000 μg, with a total dosage of up to about 1 g, depending on the route of administration. Guidance as to particular dosages or delivery methods can be found in the literature commonly available to practitioners in the art. Those skilled in the art will employ different dosage forms for nucleotides than for proteins and inhibitors. Similarly, the mode of delivery of a polynucleotide or polypeptide will depend on the particular cell, condition, location, and the like.

Diagnosis In another embodiment, an antibody that specifically binds to HRGP may be used for diagnosis of a condition or disease characterized by HRGP expression, or for HRGP or an HRGP agonist,
It can be used in assays for monitoring patients being treated with antagonists or inhibitors. Antibodies useful for diagnosis can be made in the same manner as for therapeutics described above. HRGP diagnostic assays include the use of antibodies or labels to detect HRGP in extracts of human body fluids, cells or tissues. The antibody may be used with or without modification, and may be labeled by binding to the reporter molecule either covalently or non-covalently. For some of them, various reporter molecules described above are known and can be used.

For example, ELISA (enzyme-linked immunoassay), RIA (radioimmunoassay)
As well as various protocols for measuring HRGP, such as FACS (Fluorescent Display Cell Sorter), are well known in the art, and provide the basis for diagnosing alterations or abnormalities in HRGP expression. The normal value of the expression of HRGP, that is, the standard value, is that a body fluid or a cell extract obtained from a normal subject of a mammal, preferably a human, and an antibody of HRGP are bound under conditions suitable for complex formation. Establish by. The standard complex formation can be quantified by various methods, preferably by using photometric means. The amount of HRGP expressed in the affected tissue sample and the control sample of the subject's biopsy tissue is compared to a standard value. A parameter for diagnosing the disease is established from the deviation between the standard value and the value of the subject.

In another embodiment of the invention, a polynucleotide encoding HRGP may be used for diagnostic purposes. Polynucleotides that can be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNA. This polynucleotide is used to detect and quantify gene expression in biopsy tissue where HRGP expression may be related to disease. Diagnostic assays are used to distinguish whether HRGP is present, absent, or overexpressed, and to monitor regulation of HRGP levels in therapeutic intervention. be able to.

In one embodiment, identifying a nucleic acid sequence encoding HRGP utilizing hybridization with a PCR probe capable of detecting a polynucleotide sequence, including genomic sequences, encoding HRGP or closely related molecules. Can be. The specificity of the probe, ie, whether the probe is in a very highly specific region (eg, 10 unique nucleotides in the 5 ′ regulatory region) or in a region of low specificity (eg, especially the 3 ′ coding region). Whether the probe identifies only naturally occurring HRGPs, or alleles and related sequences, depending on their origin and the stringency of the hybridization or amplification (high, medium or low) Is determined.

Probes can also be used to detect closely related sequences, preferably
It should contain at least 50% nucleotides based on any sequence encoding HRGP. The hybridization probe of the present invention comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
18, including the nucleotide sequence of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 or the naturally occurring HRGP intron, promoter, and enhancer elements It can be derived from the sequence of the genome.

As a means for preparing a hybridization probe specific to DNA encoding HRGP, a method of cloning a nucleic acid sequence encoding HRGP or an HRGP derivative into a vector for producing an mRNA probe is used. is there.
Such vectors are well known and commercially available and can be used for in vitro RNA probe synthesis by adding an appropriate RNA polymerase or an appropriate labeled nucleotide. Hybridization probes can be labeled with various reporter molecules. For example, it can be labeled with a radionuclide such as ³² P or ³⁵ S with an enzyme label such as alkaline phosphatase which binds to the probe via an avidin / biotin binding system.

The polynucleotide sequence encoding HRGP can be used for the diagnosis of a condition, disease, or disorder associated with either increased or decreased expression of HRGP. Examples of such conditions, diseases, or diseases include, but are not limited to, cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma;
Adrenal gland, bladder, bone, bone marrow, brain, breast, cartilage, cervix, gallbladder, ganglion, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary gland, skin, Cancer of the spleen, testis, thymus, thyroid, and uterus; for example, restlessness, Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatony, brain neoplasm, dementia, depression, Down's syndrome, slowness Neurological disorders such as onset dyskinesia, dystonia, epilepsy, Huntington's disease, multiple sclerosis, neurofibromatosis, Parkinson's disease, paranoid psychosis, schizophrenia, and Tourette syndrome group; and AIDS, Addison's disease, adult respiration Distress syndrome, allergy, anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes, emphysema, atrophic Inflammation, nephritis, gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocarditis or pericarditis, osteoarthritis, osteoporosis, pancreatitis, Immune responses associated with diseases such as polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, and autoimmune thyroiditis. A polynucleotide sequence encoding HRGP,
Southern or Northern blot, dot-blot or other membrane-based techniques using patient biopsy tissues or body fluids, PCR techniques, dipstick assays (test strips), pinning techniques, and Changes in HRGP expression can be detected by using ELISA assays or microarrays. Such qualitative or quantitative test methods are well known in the art.

In certain embodiments, nucleotide sequences encoding HRGP may be useful in assays that detect the activation or induction of various cancers, particularly those listed above. The nucleotide sequence encoding HRGP can be labeled by standard methods and added to a patient's body fluid or tissue sample under conditions suitable for forming a hybridization complex. After an appropriate incubation time, the sample is washed and the signal is quantified and compared to a standard value. If the amount of signal in the biopsy or extracted sample is significantly different from the signal amount in the comparable control sample, then the nucleotide sequence has hybridized to the nucleotide sequence of the sample and encodes HRGP in the sample. The occurrence of a change in the level of the nucleotide sequence indicates the presence of the associated disease. Such assays can also be used in animal studies, clinical trials, or in monitoring the treatment of an individual patient to assess the effectiveness of a particular therapeutic treatment.

To establish a basis for the diagnosis of diseases associated with HRGP expression, a normal, ie, standard, expression profile is established. This standard profile is established by combining extracts of body fluids or cells from normal subjects, either animals or humans, with HRGP-encoding sequences or fragments thereof under conditions suitable for hybridization or amplification. I can do it. The amount of standard hybridization can be quantified by comparing the values obtained in experiments using known amounts of substantially purified HRGP with those obtained in normal subjects. Standard values obtained from a normal sample can be compared to values obtained from a sample of a patient exhibiting symptoms of the disease. The presence of the disease is confirmed using the deviation between the standard value and the subject value.

Once the disease has been confirmed and the treatment protocol has begun, hybridization assays are performed periodically to determine whether the level of expression in the patient has begun to approach that observed in normal patients. Can be evaluated. The results from continuous assays can be used to determine the efficacy of treatment over a period of days or months.

For cancer, the relatively high abundance of transcripts in patient biopsies indicates a predisposition to the development of the disease. In other words, it can be a means of detecting a disease before actual clinical symptoms appear. This type of definitive diagnosis allows healthcare professionals to take preventive measures or initiate aggressive treatment earlier and prevent the onset and further progression of cancer.

Another diagnostic use of oligonucleotides designed from sequences encoding HRGP may be in PCR. Such oligomers can be chemically synthesized, made using enzymes, or made in vitro. Oligomers are preferably two nucleotide sequences used under optimal conditions to identify a particular gene or state, namely the nucleotide sequence in the sense direction (5 '→ 3') and the antisense direction (3 '← 5 '). Also, two identical or nested oligomer sets or degenerate pools of oligomers can be used under low stringency conditions to detect and / or quantify closely related DNA or RNA sequences. it can.

Methods that can be used to quantify HRGP expression include radiolabeling (
radiolabeling or biotin-labeled nucleotides, the use of co-amplification of control nucleic acids, and the use of standard graph curves drawn to complement experimental results (eg, Melby, PC et al. 1993) J. Immunol. Method
s, 159: 235-44 and Duplaa, C. et al. (1993) Anal. Biochem. 229-236). For quantification of large numbers of samples, the oligomers of interest are present in multiple dilutions and can be further quantified by performing ELISA-type assays that can be quickly quantified using a spectrophotometer or colorimetrically. Speed can be increased.

In another embodiment, oligonucleotides or larger fragments from any of the polynucleotide sequences disclosed herein can be used as targets in a microarray. By using microarrays, the expression levels of many genes can be monitored simultaneously (to generate transcript images), and gene variants, mutations, and polymorphisms can be identified. This information can be used to determine gene function, understand the genetic basis of disease, diagnose disease, and develop therapeutics and monitor their activity.

In certain embodiments, the literature (eg, Chee et al. (1995) PCT application WO 95/11995, Lock
hart, DJ et al. (1996) Nat. Biotech. 14: 1675-1680 and Schena, M. et al. (1996) P
A microarray is prepared and used according to the method described in Roc. Natl. Acad. Sci. 93: 10614-10619).

Preferably, the microarray is composed of a number of unique single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNA, fixed to a solid support. The length of the oligonucleotide is preferably a length of 6 to 60 nucleotides, more preferably a length of 15 to 30 nucleotides, and most preferably a length of 20 to 25 nucleotides. . For certain types of microarrays, it may be preferable to use oligonucleotides that are only 7 to 10 nucleotides in length. Microarrays contain oligonucleotides that cover a known 5 ′ or 3 ′ sequence, continuous oligonucleotides that cover a full-length sequence, or unique oligonucleotides selected from specific regions along the length of the sequence. Can have. The polynucleotide used in the microarray may be an oligonucleotide specific to a target gene or genes whose sequence fragment is known, or one or more species common to a particular cell type, development or disease state. May be an oligonucleotide specific to the unidentified cDNA.

To generate oligonucleotides against known sequences for microarrays, a computer algorithm is used to examine the gene of interest from the 5 ′ or more preferably the 3 ′ end of the nucleotide sequence. The algorithm identifies oligomers of defined length that have a GC content that is unique to the gene and that is within the range suitable for hybridization, and that have no putative secondary structure that could interfere with hybridization. In some cases, oligomers are synthesized at predetermined regions on the substrate using a light irradiation chemical process. The substrate can be a paper, nylon, or other type of membrane, filter paper, chip, glass slide, or any other suitable solid support.

In another embodiment, the oligomer is prepared according to the literature (eg, Baldeschweiler et al. (1995) P
It can be synthesized on the surface of a substrate by using a chemical bonding procedure and an inkjet apparatus as described in CT application WO 95/251116). In another embodiment, a dot blot or slot blot (HYBRIDOT® device
("GIBCO / BRL"), using a vacuum system, thermal, UV, mechanical or chemical coupling procedures to place and bind cDNA fragments or oligonucleotides on the surface of the substrate be able to. In another embodiment, the arrays are manually fabricated or commercially available devices (slot blot or dot blot devices), materials (any suitable solid support), and mechanical (Brinkmann® Multi) devices. (Including a channel pipettor or a robotic device), and the array can include any number of oligonucleotides, for example, in the range of 2 to 1 million, which can effectively use commercially available instruments.

To analyze a sample using a microarray, polynucleotides are extracted from a biological sample. The biological sample can be obtained from bodily fluids (eg, blood, urine, saliva, sputum, gastric juice, etc.), cultured cells, biopsy tissue, or other tissue preparations. To generate probes, polynucleotides extracted from a sample are used to create nucleic acid sequences that are complementary to nucleic acids on a microarray. If the microarray consists of cDNA, antisense RNA (aRNA) is a suitable probe. Thus, in one embodiment, mRNA is used to generate cDNA, and the cDNA is used in the presence of fluorescently labeled nucleotides to generate fragments, ie, oligonucleotide aRNA probes. These fluorescently labeled probes are incubated with the microarray such that the probe sequences hybridize to the microarray cDNA oligonucleotides. In another embodiment, a complementary nucleic acid sequence is used as a probe. This complementary nucleic acid sequence includes restriction enzymes, PCR technology, and Oligolav well known in the art of hybridization technology.
Also included are polynucleotides, fragments, and complementary or antisense sequences generated using eling or TransProbe kits (Pharmacia).

Hybridization conditions are adjusted so that hybridization occurs with exact complementary matches or with various lower levels of complementarity. After removing the hybridized probe, the level and pattern of fluorescence is determined using a scanner. The scanned image is examined to determine the degree of complementarity and relative amount of each oligonucleotide sequence on the microarray. By using a detection system, the absence, presence, and amount of hybridization can be measured simultaneously for all individual sequences. This data can be used to study large-scale interactions for sequences, mutations, variants, or polymorphisms in a sample (eg, Heller, RA et al. (1997)
Proc. Natl. Acad. Sci. 94: 2150-55).

In another embodiment of the invention, nucleic acid sequences encoding HRGP may be used to create useful hybridization probes for naturally occurring genomic sequence mapping. This sequence may map to a particular chromosome, a particular region of the chromosome, or an artificial chromosome construct. Examples of artificial chromosome products include human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BAC).
s), bacterial P1 constructs or monochromosomal cDNA libraries (eg,
Price, CM (1993) Blood Rev. 7: 127-134, and Trask, BJ (1991) Trends G
enet. 7: 149-154).

Fluorescence in situ hybridization (FISH) may have implications for other physical chromosome mapping techniques and genetic map data (eg, Verma et al. (1988) Human Chromosomes: A Manual of Basic Technique , Pergamon Press). , New York, NY ”). Examples of genetic map data can be found in various scientific journals and Online Mendelian Inher.
It can be seen in itance in Man (OMIM). With the help of correlating the location of the HRGP-encoding sequence on the physical chromosomal map with a particular disease (or predisposition to a particular disease), regions of DNA associated with a certain genetic disease can be determined. . The nucleotide sequences of the present invention can be used to detect differences in the gene sequence of normals, carriers, or patients.

[0215] Physical mapping techniques, such as in situ hybridization of chromosomal preparations and linkage analysis using established chromosomal markers, can be used to enlarge the genetic map. In many cases, even if the number or arm of a particular human chromosome is unknown, the gene arrangement on the chromosome of another mammal, such as a mouse, will reveal the relevant marker. New sequences can be assigned to chromosomal arms or parts thereof by physical mapping. This can provide valuable information to researchers investigating disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome has been localized to a particular genomic region, for example, as ataxia-telangiectasia (AT) has been located at 11q22-23 (see, eg, Gatti, RA et al. (1988) Nature 336: 577-580). Once coarsely located, any sequence that maps to that region could represent a relevant gene for further study, or a regulatory gene. The nucleotide sequences of the present invention can also be used to detect differences in chromosomal location between normals, carriers, and patients, caused by translocations, inversions, and the like.

In another embodiment of the present invention, HRGP, or a catalytic or immunogenic fragment thereof,
Oligopeptides can be used for screening compounds in various drug screening techniques. Fragments used in such screening may be in free form in solution, in a form attached to a solid support, in a form attached to a cell surface, or in a cell. Can be The binding complex formation between HRGP and the drug under test can be measured.

[0217] Alternative drug screening techniques allow for high-throughput screening of compounds with appropriate binding affinity for the protein of interest (
See, for example, Geysen et al. (1984) PCT application WO 84/03564). In this method, HRGP
When applied to a large number of different small test compounds, they are synthesized on a solid substrate such as a plastic pin or other surface. The compound to be tested was designated as HRGP
Or react with fragments thereof and wash. The bound HRGP is then detected by methods well known in the art. Also, purified HRGP can be directly coated on a plate for use in the aforementioned drug screening techniques. Alternatively, the peptide can be captured using a non-neutralizing antibody and immobilized on a solid substrate.

In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of binding to HRGP specifically compete with the compound under test for binding to HRGP. In this method, antibodies can be used to detect the presence of any peptide that has one or more antigenic determinants of HRGP in common.

In yet another embodiment, even in molecular biology techniques that have not been developed to date, the new techniques may have properties known to date for nucleotide sequences (eg,
HRGP-encoding nucleotide sequences can be used in techniques based on, but not limited to, the triplet genetic code and properties such as specific base pairing.

The following examples are merely illustrative of the present invention and are not intended to limit the present invention to these examples.

[0221]

【Example】

For illustrative purposes, Insight clone No. 1913206 was used to isolate the PRO
The preparation and sequencing of the STUT04 cDNA library will be described. Preparation of cDNA libraries in LIFESEQ ^TM database and sequencing has been changing over time, the use of a particular time available kits, plasmids, and device library was produced and analyzed changes gradually .

1. Preparation of PROSTUT04 cDNA Library A PROSTUT04 cDNA library was prepared from a 57-year-old Caucasian male prostate cancer tissue. Surgery included radical prostatectomy, removal of both testicles, and removal of regional lymph nodes. Pathology reports indicated adenocarcinoma (Gleason grade 3 + 3) around both the left and right prostate. Perineural infiltrates were present and extended to periprostatic tissue. One of the right pelvic lymph nodes, left and right surgical margins, was tumor-positive and seminal vesicles were negative. The patient's history included tonsillectomy with adenoidectomy, appendectomy, and benign neoplasms of the large intestine. The patient had been taking insulin for Type I diabetes. The patient's family history included a malignant neoplasm of the patient's father's prostate and type I diabetes without maternal complications.

[0223] Frozen tissues were purified using a Brinkmann homogenizer POLYTRON-PT-3000 (Brinkmann Instrum).
ents, Westbury NY) and homogenized in guanidine isothiocyanate solution. According to Stratagene's RNA isolation protocol (Stratagene), phenol extracted with chloroform pH 5.5 and then 1.0 ml of 2M sodium acetate was added to the lysate extracted with acidic phenol pH 4.7. RNA was precipitated twice with an equal volume of isopropanol according to the Stratagene protocol. The RNA pellet was resuspended in DEPC-treated water and treated with DNase at 37 ° C for 50 minutes. The reaction was stopped with an equal amount of acidic phenol. RNA was precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol and resuspended in DEPC-treated water. Qi RNA
It was isolated using the agen Oligotex kit (QIAGEN Inc, Chatworth, CA) and used to create a cDNA library.

[0224] This RNA was prepared using the SuperScript Plasmid System for cDNA synthesis and plasmi.
Handled according to d cloning (p / n 18248-013, Gibco / BRL) recommended protocol. The cDNA was fractionated on a Sepharose CL4B column (Part No. 275105-01; Pharmacia) and the cDNA over 400 bp was ligated to pSportI. Next, this plasmid pSpo
rtI was transformed into DH5α ^™ competent cells (Part No. 18258-012; Gibco / BRL).

2 Isolation and Sequencing of cDNA Clones Plasmid DNA was released from the cells, and this was purified using the REAL Prep 96 Plasmid Kit.
(Catalog # 26173, QIAGEN, Inc., Chatsworth, CA). This kit uses a multi-channel reagent dispenser for 96-well blocks.
This enabled simultaneous amplification of samples. I used the recommended protocol,
The following points have been changed. (1) Bacteria were cultured in 1 ml of sterile Terrific Broth (Part No. 22711, Gibco / BRL) containing 25 mg / L carbenicillin and 0.4% glycerol. (2) After inoculation, the culture solution was incubated for 19 hours, and at the end of the incubation, the cells were lysed in 0.3 ml of lysis buffer. (3) After isopropanol precipitation, the pellet of plasmid DNA
Resuspended in 1 ml of distilled water. After the last step of the protocol, 96 samples
Transferred to a hole block and stored at 4 ° C.

The sequencing of this cDNA was performed using Peltier Thermal Cyclers (MJ Research, Water
town, MA PTC200) and Applied Biosystems 377 DNA Sequencing System
Using Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with ms
The reading frame was determined by the method of Sanger et al. (1975, J. Mol. Biol. 94: 441f).

3 Using the nucleotide sequences of the homology search sequence listing of cDNA clones and their inferred proteins and the amino acid sequences inferred therefrom as query sequences, for example, GenBank, SwissProt, BLOCKS, and Pima II I searched the database. These databases contain annotated sequences that have already been identified, and a region having homology (similarity) was searched from this database using BLAST (for Basic Local Alignment Tool) (Al
tschul. SF (1993) J. Mol. Evol. 36: 290-300; Altschul et al. (1990) J. Mol. B
iol. 215: 403-10).

BLAST creates an alignment of both nucleotide and amino acid sequences to determine sequence similarity. Due to the local nature of the alignment, BLAST is particularly useful in identifying exact matches, ie, homologs of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. Other algorithms can be used to handle primary sequence patterns and gap penalties for secondary structure (eg, as described in Smith, T. et al. (1992) Protein Engineering 5: 35-51). The sequences disclosed herein are at least 49 nucleotides in length and have no more than 12% of unnecessary bases (where N is recorded other than A, C, G, or T). .

The BLAST method searches for matches between the query and database sequences, evaluates the statistical significance of any matches found, and reports only matches that meet a user-selected significance threshold. . The threshold in this application was set to 10 ^{-25 for} nucleotides and 10 ^-14 for peptides.

The nucleotide sequence of Incyte was searched in the GenBank database of primates (pri), rodents (rod), and other mammalian sequences (mam), and the amino acid sequence deduced from the same clone was then retrieved. , GenBank Functional Protein Database, Mammals (mamp), Vertebrate (vrtp), and Eukaryotes (eukp).

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of transcripts of a gene, where a labeled nucleotide sequence and the RN of a particular cell type or tissue are
Hybridization with the membrane to which A is bound (Sambrook et al.,
Supra).

Similar computer techniques using BLAST are used to search for identical or related molecules in databases such as GenBank or LIFESEQ ^™ database (Incyte Pharmaceuticals). This analysis can be performed in a much shorter time than hybridization using many membranes. In addition, the sensitivity of the computer search can be modified to determine whether a particular match is classified as an exact match or homologously.

[0233] The reference value of the search is a product score, which is defined by the following equation. (Sequence match (%) × Maximum BLAST score (%)) / 100 This product score takes into account both the degree of similarity between two sequences and the match in sequence length. For example, when the product score is 40, the match is accurate within an error of 1-2%, and when the score is 70, the match is exact. Homologous molecules are usually identified by selecting those that show a product score of 15-40, while those with lower scores are identified as closely related molecules.

The results of the Northern analysis are reported as a list of libraries in which transcripts encoding HRGP are generated. A list of sequence abundance and percent abundance is also reported. Abundance directly reflects the number of detections of a particular transcript, and percent abundance is the abundance divided by the total number of sequences detected in the cDNA library.

Extension of HRGP-encoding sequence One sequence of the polynucleotide of the invention was used to design oligonucleotide primers for extending a partial nucleotide sequence to full length. One primer was synthesized to initiate extension in the antisense direction and the other primer was synthesized to extend the sequence in the sense direction. Using these primers, the known HRGP sequence was extended "outside" to generate an amplicon containing a new, unknown nucleotide sequence of the region of interest. The first primer is OLIGO 4.
06 (National Biosciences), or other suitable program, with a length of about 22 to about 30 nucleotides and 50% or more G
It was designed to have a C content and anneal to the target sequence at a temperature of about 68 to about 72 ° C. Stretches of any nucleotides that would result in hairpin structure and primer-primer dimerization were excluded.

This sequence was extended using a selected human cDNA library (Gibco / BRL). If more than one step extension is necessary or desirable, an additional set of primers is designed to further extend the known region.

High fidelity amplification is achieved by thorough mixing of the enzyme and reaction mixture according to the instructions in the instructions for the XL-PCR kit (Perkin Elmer). When starting amplification with 40 pmol of each primer and all other components of the kit at the recommended concentration, the following parameters were used using a Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA), ie, step 194: 1 minute at 65 ° C (initial denaturation) Step 2 1 minute at 65 ° C Step 3 6 minutes at 68 ° C Step 4 15 seconds at 94 ° C Step 5 1 minute at 65 ° C Step 6 7 minutes at 68 ° C Step 7 Steps 4-6 Repeat 15 more cycles Step 8 94 ° C for 15 seconds Step 9 65 ° C for 1 minute Step 10 68 ° C for 7 minutes 15 seconds Step 11 Repeat steps 8 to 12 for 12 cycles Step 12 72 ° C for 8 minutes Step 134 ° C (the (The temperature was maintained).

An aliquot of 5-10 μl of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose minigel to determine whether the sequence extension reaction was successful. Were determined. Select the largest product or band, cut out of the gel,
Purification was performed using QIAQuick ^™ (QIAGEN Inc., Chatsworth, CA) and the single-stranded nucleotide overhang was excised using the Klenow enzyme to create blunt ends that facilitate recombination and cloning.

After ethanol precipitation, the product was redissolved in 13 μl ligation buffer and 1 μl
Of T4-DNA ligase (15 units) and 1 μl of T4 polynucleotide kinase were added, and the mixture was incubated for 2-3 hours at room temperature or overnight at 16 ° C. Competent E. coli cells (in 40 μl of appropriate culture medium)
The ligation mixture was transformed with 80 μl of SOC medium (Semb
rook et al., supra). After 1 hour incubation at 37 ° C, all transformation mixtures were plated on Luria Bertani (LB) agar (Sembrook et al., Supra) containing 2xCarb. At a later date, several colonies are randomly selected from each plate,
Cultivated in 150 μl of liquid LB / 2 × Carb medium in each well of a suitable commercially available sterile 96-well microtiter plate. Further at a later date, 5 μl each
Was transferred into a non-sterile 96-well plate and diluted 1:10 with water,
5 μl of each sample was transferred to a PCR array.

For PCR amplification, 18 μl of the enriched PCR reaction mixture (3.3 ×) containing 4 units of rTth DNA polymerase, vector primers, and / or primers specific to the gene used in the extension reaction were added to each well. Added. The amplification was carried out under the following conditions: Step 1 at 94 ° C. for 60 seconds Step 2 at 94 ° C. for 20 seconds Step 3 at 55 ° C. for 30 seconds Step 4 at 72 ° C. for 90 seconds Step 5 Steps 2 to 4 were further repeated 29 cycles Step 6 72 Performed at 74 ° C. (maintained) at 180 ° C. for 180 seconds.

An aliquot of the PCR reaction was run on an agarose gel with molecular weight markers. The size of the PCR product was compared to the original partial cDNA, the appropriate clone was selected, ligated to the plasmid and sequenced.

Similarly, using one of the nucleotide sequences of the invention, the 5 ′
It is possible to obtain regulatory sequences, obtain oligonucleotides designed for extension in the 5 'direction, and obtain appropriate genomic libraries.

6 Labeling and Use of Hybridization Probes cDNA, mRNA and genomic DNA are screened using hybridization probes made from one of the nucleotide sequences of the present invention. Particular mention is made of the labeling of oligonucleotides consisting of about 20 base pairs, but generally the same procedure is used for larger cDNA fragments. OLIGO4.06 (Nationa
Oligonucleotides were designed using state-of-the-art software such as Bioscience) and 50 pmol of each oligomer, 250 μCi of [γ- ³² P] adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN, Bo
ston MA). The labeled oligonucleotide is purified using a Sephadex G-25 ultrafine resin column (Pharmacia & Upjohn). Aliquots containing the labeled probe at 10 ⁷ counts per minute are applied to a common membrane of human genomic DNA digested with one of the endonucleases AseI, BglII, EcoRI, PstI, Xba1 or PvuII; DuPont NEN. Used in hybridization analysis.

The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH).
Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, blots are washed sequentially at room temperature under stepwise increasing stringency conditions up to 0.1 × aqueous sodium citrate and 0.5% sodium dodecyl sulfate. After exposing the XOMAT AR ^™ film (Kodak, Rochester, NY) to the blot in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale, CA) for several hours, the hybridization patterns are visually compared.

7 Microarrays To make oligonucleotides for microarrays , SEQ ID NO: 2 was
The nucleotide sequence is examined using a computer algorithm from the 3 'end. The algorithm identifies oligomers of defined length that are unique to the gene, have a GC content within the range suitable for hybridization, and are free of putative secondary structures that would prevent hybridization. . This algorithm identifies 20 sequence-specific oligonucleotides 20 nucleotides in length (20 mer). Only one central nucleotide in each sequence is changed to create a set of otherwise identical oligonucleotides. This process is repeated for each gene in the microarray, and two sets of 20 20-mers are synthesized and arranged on the surface of a silicon chip using a light-irradiated chemistry process (Chee, M. et al., PCT / WO95). / 11995).

Alternatively, oligomers are synthesized on the surface of a substrate using a chemical coupling procedure and an inkjet device (Baldeschweiler, JD et al. PCT / WO95 / 25116). Yet another method uses a "lattice-type" array similar to that used in dot blots (slot blots), using a vacuum system, thermal, UV, mechanical or chemical binding procedures, and cDNA fragments. Alternatively, the oligonucleotide is placed on the surface of the substrate and allowed to bind. Arrays can be fabricated manually or by using commercially available materials and machines, and can include any suitable number of fragments or oligonucleotides. After hybridization, the microarray is washed to remove unhybridized probes, and the level and pattern of fluorescence are determined using a scanner. The scanned images are examined to determine the degree of complementarity and the relative amount / expression level of each oligonucleotide sequence on the microarray.

8 Complementary Polynucleotides The sequence encoding HRGP, or a sequence complementary to any portion thereof, is used to reduce, ie, inhibit, the expression of naturally occurring HRGP. Although the use of oligonucleotides of about 15 to about 30 base pairs is specifically noted, essentially the same procedure can be used for smaller or larger sequence fragments.
An appropriate oligonucleotide is designed using the Oligo 4.06 software and the sequence of any one of the nucleotides of the present invention. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and used to prevent promoter binding. To inhibit translation, a complementary oligonucleotide is designed so that the ribosome does not bind to the transcript encoding HRGP.

9 HRGP Expression HRGP expression is achieved by subcloning the cDNA into a suitable vector and transfecting the vector into a host cell. In this case, a cloning vector is also used to express HRGP in E. coli. This vector has a β-galactosidase promoter upstream of the cloning site, followed by an amino-terminal Met, followed by seven residues of β-galactosidase. Following these eight residues are a bacteriophage promoter that facilitates transcription and a linker that contains many unique restriction sites.

The transformed strain was isolated, induced with IPTG in a standard manner, and fused with the first 7 residues of β-galactosidase, a linker of about 5 to 15 residues, and a full-length protein. Produce protein. This group of signal residues induces secretion of HRGP into the bacterial growth medium, which can be used directly in subsequent assays for activity.

Generation of 10 HRGP-Specific Antibodies Using standard protocols, immunize rabbits with HRGP substantially purified using PAGE electrophoresis (Sambrook, supra) to produce antibodies. The amino acid sequence deduced from one of the nucleotide sequences of the present invention is analyzed using DNASTAR software (DNASTAR Inc.) to determine a region with high immunogenicity, to synthesize the corresponding oligopeptide, and to those skilled in the art. This is used to raise antibodies in a well-known manner. Techniques for selecting appropriate peptide sequences and producing antibodies are well known to those skilled in the art. The selection of suitable epitopes, such as epitopes near the C-terminus or in hydrophilic regions, is described in Ausubel et al., Supra, and other references.

Generally, an oligopeptide having a length of 15 residues is synthesized by chemistry of the fmoc method using a peptide synthesizer Model 431A of Applied Biosystems, and M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS
: Keyhole limpet hemocyanin (KLH, Sigma, St. Louis, MO) by reaction using Ausubel et al., Supra. Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. To test the anti-peptide activity of the obtained antiserum, for example, the peptide is bound to plastic,
Block with 1% BSA, react with rabbit antiserum, wash, and react with goat anti-rabbit IgG labeled with radioactive iodine.

11 Purification of naturally occurring HRGP using specific antibody Naturally occurring HRGP or recombinant HRGP is substantially purified by immunoaffinity chromatography using an antibody specific for HRGP. An immunoaffinity column is constructed by covalently linking an HRGP antibody to an activated chromatography resin such as CnBr-activated Sepharose (Pharmacia & Upjohn). After binding, the resin is blocked and washed according to the instructions for use.

The culture solution containing HRGP is passed through an immunoaffinity column, and the column is
Wash under conditions that allow RGP to be preferentially adsorbed (eg, with a high ionic strength buffer in the presence of a surfactant) The column is eluted under conditions that disrupt the binding of the antibody to HRGP (eg, a buffer of pH 2-3 or a high concentration of chaotropic ions such as urea or thiocyanate ions) to recover HRGP.

12 Identification of Molecules That Interact with HRGP HRGP, or a biologically active fragment thereof, was prepared using ¹²⁵ I Bolton Hunter reagent (Bol
(1973) Biochem. J. 133: 529). Candidate molecules pre-sequenced in the wells of the multiwell plate are incubated with labeled HRGP, washed, and the wells bearing the labeled HRGP complex are assayed. Using the data obtained using different concentrations of HRGP, the association, affinity, and number values of the candidate molecule and HRGP are calculated.

All publications and patent specifications mentioned above are incorporated herein by reference.
Various modifications of the method and system of the invention described herein which do not depart from the scope and spirit of the invention will be apparent to those skilled in the art. Although the invention has been described with reference to particularly preferred embodiments, it should be understood that the true scope of the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/47 C12N 1/15 4C084 16/18 1/19 4H045 C12M 1/00 1/21 C12N 1/15 C12P 21/02 C 1/19 C12Q 1/68 A 1/21 C12N 15/00 ZNAA 5/10 A61K 37/02 C12P 21/02 C12N 5/00 A C12Q 1/68 F (81) Designated country EP (AT , BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM) , GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, Y, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, U S, UZ, VN, YU, ZW (72) Inventor Bandman, Olga 94043, Mountain View Anna Avenue, California, United States 366 (72) Inventor Hillman, Jennifer El, 94040, Mountain View, California, United States 12 Monrode Drive 230 (72) Inventor Au-Young, Janice 94702 Berkeley Caines Avenue, California, United States 1419 (72) Inventor Tang, Tom Wye 94087, California, USA Sunnyvale # 14 East Remington Drive 110 (72) Inventor Yue, Henry Sunnyvale Lewis Avenue, 94087, California, United States 826 (72) Inventor Shah, Parvi Sunnyvale # 5 Queen Charlotte Drive, 94087, California, United States 1608 (72) Inventor Gegler, Carl Jay Menlo Park Oakland Avenue, CA 94025, United States of America 1048 (72) Inventor Korley, Neil Sea CA 94040, CA, United States Mountain View # 30 Dale Avenue 1240 F-term (reference) 4B024 AA01 AA11 BA44 BA80 CA04 CA09 CA12 DA02 DA05 DA11 EA04 FA01 GA01 GA11 HA01 HA03 4B029 AA07 BB20 CC03 FA12 4B063 QA01 QA19 QQ01 QQ42 QQ52 QR08 QR42 QR56 QR59 QR62 QR80 QS05 AG01 CC25 QS05 AG01 CC24 QS05 DA01 DA13 4B065 AA01X AA57X AA87X AB01 AB02 BA02 BA08 CA24 CA25 CA44 CA46 4C084 AA02 AA07 AA17 BA01 BA08 BA13 BA22 CA18 DC50 NA14 ZB071 ZB261 4H045 AA10 AA11 BA10 CA40 DA76 EA20 EA50 FA72 FA74

Claims (20)

[Claims] 1. SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9
A substantially purified human regulatory protein (HRGP) comprising a polypeptide having an amino acid sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. 2. An isolated and purified polynucleotide that hybridizes under stringent conditions to the polynucleotide encoding HRGP of claim 1. 3. SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 2.
0, an isolated and purified polynucleotide having a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24. 4. A microarray comprising at least a fragment of at least one of the polynucleotides encoding HRGP of claim 1. 5. An isolated and purified polynucleotide having a nucleic acid sequence that is complementary to the nucleic acid sequence of the polynucleotide of claim 3. 6. A composition comprising the polynucleotide of claim 3. 7. An expression vector comprising the polynucleotide of claim 3. 8. A host cell comprising the vector of claim 7. 9. A method for producing a polypeptide encoding a human / regulatory protein, comprising: (a) culturing the host cell of claim 8 under conditions suitable for expression of the polypeptide; And b) recovering the polypeptide from the culture medium of the host cell. 10. A pharmaceutical composition comprising the substantially purified human regulatory protein of claim 1 together with a suitable pharmaceutical carrier. 11. A purified antibody that specifically binds to the human regulatory protein of claim 1. 12. A purified agonist of the human regulatory protein of claim 1. 13. A purified antagonist of the human regulatory protein of claim 1. 14. A method of stimulating cell proliferation, comprising the step of providing the cell with an effective amount of the human regulatory protein of claim 1. 15. A method for treating or preventing cancer, comprising a step of administering an effective amount of the pharmaceutical composition of claim 10 to a patient in need of such treatment. 16. A method for treating or preventing cancer, comprising administering an effective amount of the antagonist of claim 13 to a patient in need of such treatment. 17. A method of treating or preventing an immune response, comprising administering to a patient in need of such treatment an effective amount of the antagonist of claim 13. 18. A method for detecting a nucleic acid sequence encoding a human / regulatory protein in a biological sample, comprising: (a) hybridizing the polynucleotide of claim 5 with the nucleic acid sequence of the biological sample. Forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex encodes a human / regulatory protein in the biological sample. A method for detecting a nucleic acid sequence encoding a human / regulatory protein in a biological sample, comprising the steps of correlating with the presence of the nucleic acid sequence. 19. A method for detecting the expression level of a nucleic acid sequence encoding a human regulatory protein in a biological sample, comprising: (a) the nucleic acid sequence of the biological sample, and the polynucleotide of claim 5; And (b) identifying the presence of said hybridization complex, thereby allowing the presence of a nucleic acid sequence encoding a human / regulatory protein in said biological sample. Determining the expression level of a nucleic acid sequence encoding a human / regulatory protein in a biological sample. 20. The process of performing the hybridization, wherein the polynucleotide of the biological sample is amplified by polymerase chain reaction.
The method according to claim 19, wherein the labeling is performed.